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Environment International
journal homepage: www.elsevier.com/locate/envint
Integration of metabolomics and transcriptomics reveals short-chainchlorinated paraffin-induced hepatotoxicity in male Sprague-Dawley rat
a CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning 116023, ChinabUniversity of Chinese Academy of Sciences, Beijing 100049, Chinac Institute of Quality and Standard for Agro-Products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, Chinad Safety Evaluation Center of Shenyang Research Institute of Chemical Industry Ltd, Shenyang 110021, Chinae Toxicology Program and Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada
A R T I C L E I N F O
Handling editor: Yong-Guan Zhu
Dedicated to the 70th anniversary of DalianInstitute of Chemical Physics, CAS.
Background: Short-chain chlorinated paraffins (SCCPs) used in various industrial applications have been listed asnew POPs. Previous studies based on high-dose exposures indicate their hepatotoxicity. However, their me-chanisms of toxicity or adverse outcome pathways and health risks remain largely unknown.Objectives: This study aimed to evaluate metabolic consequences of chronic dietary exposure to SCCPs at lowdoses and reveal the molecular mechanisms underlying hepatotoxicity of SCCPs.Methods: A combination of transcriptomics and metabolomics, together with general pathophysiological testswere performed to assess the hepatic response of male rats exposed to SCCPs.Results: Our results highlight two major modes of action: Inhibition of energy metabolism and activation of theperoxisome proliferator-activated receptor α (PPARα). Exposure to SCCPs suppressed oxidative phosphoryla-tion, glycolysis, gluconeogenesis and turnover of ATP-ADP-AMP and thus results in deficiencies of amino acidsand nucleotides in liver of the rat. Exposure to SCCPs affected expression levels of 13 genes downstream ofPPARα that encode proteins associated with metabolism of fatty acids. As a result, peroxisomal and mi-tochondrial fatty acid β-oxidation, microsomal fatty acid ω-oxidation, and lipogenesis were accelerated.Conclusions: Results of this work strongly support the conclusion that low-dose exposure to SCCPs can result inadverse outcomes in the rat model. Significant SCCP-induced inhibition of energy metabolism occurs at en-vironmentally relevant dosages, which suggests that SCCPs exhibit metabolic toxicity. Interactions of SCCPs withPPARα signaling pathway can explain the disruption of lipids and amino acids metabolism.
1. Introduction
Short-chain chlorinated paraffins (SCCPs) are mixtures of poly-chlorinated n-alkanes with 10–13 carbon atoms. As a constituent ofchlorinated paraffins, SCCPs are widely used in various industrial ap-plications, including plasticizers, flame retardants, metal-workingfluids, lubricant additives, paints and sealants (Fiedler, 2010). Due totheir environmental persistence, bioaccumulation potential, long-rangetransport potential and adverse health effects, in 2017, SCCPs werelisted in Annex A of the Stockholm Convention on Persistent OrganicPollutants (POPs) (Ali and Legler, 2010; Hüttig and Oehme, 2005; Liet al., 2016; Wei et al., 2016). Annual production of SCCPs worldwidein 2016 was estimated to exceed 165,000 tons (Gluge et al., 2016). As a
result of the production and the wide variety of industrial applications,SCCPs have been ubiquitously detected in environmental matrixes andhuman body (Wei et al., 2016; Thomas et al., 2006; Xia et al., 2017; Liet al., 2017), posing potential hazards to wildlife and humans.
SCCPs exhibit little potency for acute toxicity in animals but arepotentially carcinogenic (Bucher et al., 1987). In sub-chronic studies oftoxicity to rats and mice, SCCPs produced toxic effects on liver, kidney,thyroid, and parathyroid glands (Nielsen and Ladefoged, 2013). The no-and lowest-observed adverse effect levels (NOAEL and LOAEL) of SCCPswere 10 and 100mg/kg body mass (bm)/d for rats, respectively (UNEP,2011). Exposure to SCCPs results in up-regulated expression of somecytochrome P450 family genes (Zhang et al., 2016), which alters in-tracellular redox status (Geng et al., 2015). SCCPs have been reported
https://doi.org/10.1016/j.envint.2019.105231Received 26 July 2019; Received in revised form 24 September 2019; Accepted 27 September 2019
⁎ Corresponding authors at: Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China.E-mail addresses: [email protected] (H. Zhang), [email protected] (J. Chen).
to induce peroxisome proliferation, which is associated with damage toliver (Wyatt et al., 1993). Moreover, SCCPs function as endocrine dis-ruptors. Based on results observed during in vivo studies, exposure toSCCPs reduces concentrations of free thyroid hormone and disruptsthyroid signaling, although the thyroid gland is not a direct target ofSCCPs (Liu et al., 2016; Gong et al., 2018). Additionally, a recent in vitrostudy has also shown that SCCPs not only exert their endocrine-dis-rupting effects through estrogen receptor α and the glucocorticoid re-ceptor, but also alter 17 β-estradiol and release of cortisol (Zhang et al.,2016). These endocrine-disrupting effects, together with oxidativestress, imply that SCCPs might cause adverse effects to health, even atlow doses. Currently data are insufficient to comprehensively assess thebiological events caused by SCCPs, and very limited information isavailable regarding their toxic effects at environmentally relevant do-sages or internal concentrations in tissues of humans.
The correlation between exposure to endocrine disruptors and theireffects on health is often difficult to determine, mainly due to the oc-currence of low-dose effects and non-monotonic dose-responses(Vandenberg et al., 2012). However, the advent of omics technologieshas made these analyses possible. Transcriptomics identifies broad-scale changes in expression levels of genes, which might help re-searchers ascertain specific, molecular responses as endpoints of low-dose effects and lead to the generation of hypotheses about the me-chanisms of action (Shi et al., 2006). Metabolomics can reveal system-wide alterations in metabolic pathways in response to low-dose ex-posure and facilitate identification of early biomarkers of a toxicant(Spivey, 2004). It was hypothesized that SCCPs initiate specific biolo-gical events and disturb global metabolism at exposures relevant toconcentrations observed in tissues of humans or environmental ma-trices. The present study was designed to test this hypothesis in the liverof male Sprague-Dawley (SD) rats using a combination of tran-scriptomic and metabolomic approaches.
In this study, exposure to 0.01mg SCCP/kg body mass/day (kg bm/d) was selected as low dose, which is comparable to the human dailyexposure level of SCCPs, specifically via infant lactational exposure:0.34–8.65 μg/kg bm/d (Cao et al., 2017; Xia et al., 2017), inhalationexposure:< 0.01–5.9 μg/kg bm/d (Friden et al., 2011; Shi et al., 2017),dietary exposure: 0.32–1.1 μg/kg bm/d (Cao et al., 2015; Gao et al.,2017), occupational exposure: 40.7, 22.6, and 62.2 μg/kg bm/d forworkers, local adults and children in a mega e-waste recycling in-dustrial park with high end exposure (Chen et al., 2018). The dose of1mg/kg bm/d was selected as middle dose. The LOAEL of 100mg/kg bm/d was adopted as a positive control dose (high dose) to link thelow-dose effects with the known deleterious outcomes of SCCP ex-posure. The hepatic responses of male SD rats exposure to SCCPs werecharacterized by use of transcriptomics and metabolomics, togetherwith general pathophysiological endpoints. The goal was to identifymultiple targets affected by SCCPs and to determine some early re-sponses.
2. Methods
2.1. Chemicals
SCCPs used in the present study were a mixture of C10–13-CPs(chlorine content: 56.5% by mass; purity:> 99.9%) synthesized bychlorination of C10–13-n-alkanes (C10:C11:C12:C13= 1:1:1:1, mass ratio)in our laboratory. The chromatographic profile of prepared SCCPs wassimilar to that of a SCCP standard (55.5% Cl) obtained from Dr.Ehrenstorfer (Augsburg, Germany) (Fig. S1). HPLC-grade acetonitrile(ACN) and methanol were obtained from Fisher (Fair Lawn, NJ, USA).Ammonium acetate and formic acid were obtained from J&K Scientific(Beijing, China). The water used in all experiments was ultrapure waterfrom a Milli-Q system (Millipore, Billerica, MA, USA). Six internalstandards (1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine,octanoyl (8,8,8-D3)-L-carnitine, 1-lauroyl-2-hydroxy-sn-glycero-3-
phosphocholine, hendecanoic acid, L-phenylalanine-d5 and non-adecanoic acid) were purchased from Sigma-Aldrich (Sigma ChemicalCo, USA). Internal standards were dissolved into methanol before me-tabolites extraction.
2.2. Animal experiment
All animal experiments were performed according to protocols ap-proved by the Institutional Animal Care and Use Committee in theSafety Evaluation Center, Shenyang Research Institute of ChemicalIndustry, China. Male SD rats (8 weeks old; with weight ranging from339 to 407 g) were housed in a controlled environment at a tempera-ture of 25 ± 3 °C on a 12-h light/dark cycle. After 5 d of acclimation,rats were randomly assigned to one of four groups, each containing tenanimals. SCCPs were administered to rats at various doses (0, 0.01, 1and 100mg/kg bm/d) for 28 d. In the exposure groups, SCCPs weredissolved in corn oil and administered by gavage, while the controlgroup received corn oil only. Body mass was monitored and recordedevery 4 d throughout the experiment. Rats were euthanized humanelyunder diethyl ether anesthesia after 28 days of exposure. Blood andurine were collected for clinical chemistry and hematology analyses.Livers were surgically removed and weighed. A fraction of the liverswas fixed with a 10% formalin solution containing neutral phosphate-buffered saline for the histopathological examination; the remainingtissues were frozen in liquid nitrogen and stored at –80 °C for thequantification of gene expression and metabolite levels.
2.3. Pathophysiological test
Sixty-one hematological and biochemical parameters were mea-sured using an automated hematology analyzer (Sysmex XT-2000i,Japan), a biochemical blood analyzer (Hitachi 7180, Hitachi, Japan)and an automated urine analyzer (Uritest-500B, China). The in-vestigated hematological and biochemical parameters are shown inTables S1–3. Liver tissues were embedded in paraffin, stained withhematoxylin and eosin, and examined under a light microscope (AxioImager A1, Germany).
2.4. Exposure estimation
SCCPs were measured in liver collected after 28 days of oral ad-ministration. Samples were analyzed using previously publishedmethods (Gao et al., 2011) that involved extraction, cleanup and finaldetermination performed on a trace gas chromatograph coupled to aTrace DSQ II mass spectrometer in ECNI mode (GC/ECNI-MS, Thermo,USA). Approximately 100mg of the liver samples were extracted withultrasonic solvent extraction, purified on a multilayer silica gel column,then evaporated and redissolved in 20 μL of the internal standard so-lution (13C6-α-HCH in n-nonane) for the instrumental analysis. Chro-matographic separation was performed with a capillary DB-5 column (J&W Scientific, USA). Three samples from each group were subjected tothe quantitative analysis, and the concentrations of SCCPs were nor-malized to mass of liver.
2.5. Transcriptomic analysis
Livers from four rats in each treatment group were randomly se-lected for transcriptomic analysis. Total RNA was isolated using themirVana™ miRNA Isolation Kit (Ambion, US) and subsequently ampli-fied and labeled with cyanine-3 (Cy3) by use of the Low Input QuickAmp Labeling Kit, the microarray analysis was performed with wholerat genome 4×44 K arrays. Briefly, each slide was hybridized with1.65 μg of Cy3-labeled cRNA for 17 h, washed in staining dishes, andscanned on an Agilent Microarray Scanner (Agilent Technologies, US).
Eight significant differentially expressed genes (DEGs) (Cdk1, Ednra,Pfkfb1, Slpi, Herpud1, Acaa1b, Alas1 and Acot2) were selected for
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validation of quantitative real-time polymerase chain reaction (qRT-PCR) using the ABI power SYBR green PCR master mix (ABI, USA).Amplification was performed using a 7900 HT Sequence DetectionSystem (ABI, USA), and a melting curve analysis was performed toconfirm specificity of qRT-PCR. Detailed information about the tran-scriptomic analysis is provided in Supplemental Material, Gene ex-pression analysis.
2.6. Metabolomic analysis
Livers from ten rats in each group were used for metabolomicsanalysis, samples of each liver (100mg) were mixed with 1mL of H2Orespectively, homogenized, and then ultrasonicated for 5min in an ice-water bath. Samples were subsequently freeze-dried and extracted witha mixture of methanol/water (4:1, v:v). The resulting solution wasvortexed for 30min and then centrifuged at 13,000g for 20min at 8 °C.Finally, the supernatant was filtered through an organic phase filter andtransferred to a vial for the metabolite analysis. Prior to extraction, sixinternal standards were spiked into the sample for quality control of thesample preparation and instrumental analysis.
A pseudotargeted approach was adopted for the metabolomic ana-lysis. This approach exhibited greater repeatability and wider linearrange than the traditional untargeted metabolomic method. Briefly, rat
liver extracts were analyzed on a Waters Acquity ultra-performanceliquid chromatography coupled online to an ABI Q-Trap 5500 (ABSCIEX, USA) system (UPLC/Q-Trap MS) operated in multiple reactionmonitoring (MRM) mode, for which the MRM ion pairs were acquiredfrom the liver extracts through untargeted tandem MS using an Agilent1290 Infinity ultra-performance liquid chromatography system coupledonline to an Agilent 6540 Q-TOF MS (Agilent, Santa Clara, CA) system(UPLC/Q-TOF MS). The same liquid chromatography conditions wereemployed for both UPLC/Q-Trap MS and UPLC/Q-TOF MS. Six hundredeighty-eight ion pairs with defined parameters were monitored. Thedetails of the instrumental analyses are shown in SupplementalMaterial, LC-MS-based metabonomics analysis.
2.7. Data processing
Statistical differences were performed by using Student's t-test forcomparisons between two groups or One-Way ANOVA for multiplecomparisons with Duncan's method. The normality assumption andvariance homogeneity tests were performed before ANOVA.Hierarchical cluster analysis (HCA) was conducted using MeV software.Principal component analysis (PCA) and partial least squares dis-criminate analysis (PLS-DA) were applied with unit variance (UV)scaling using SIMCA-P 11.5 (Umetrics, Sweden). The VIP value of every
Fig. 1. Divergence in expression levels of genes and metabolism-related alteration on transcriptional level of SD rat liver after 28-day oral administration of SCCPs.(A) PLS-DA score plot of the genes identified from the control and SCCP-treated groups. (B) TELI of liver transcription fingerprint in the control and SCCP-treatedgroups. (C) The Venn diagram shows the DEGs overlap among the three SCCP-treated groups. (D) Hierarchical clustering based on the DEGs related to metabolism. *,P < 0.05; **, P < 0.01.
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metabolite ion is also calculated by SIMCA-P.Microarray gene expression data were extracted with feature ex-
traction software 10.7 (Agilent), normalized by GeneSpring software11.0 (Agilent). Background corrected signal was used and raw datawere normalized by Quantile algorithm. MultiQuant software 3.0 (ABSCIEX) was used for metabolomic data processing of the Q-Trap MSdata. The correlation network of metabolites was constructed using theCytoscape software package (version 2.8.2). The transcriptional effectlevel index (TELI) and metabolic effect level indexes (MELI) were cal-culated to describe the dose-response behavior of SCCP exposure.Detailed calculation methods are shown in the Supplemental Material,Determination of TELI and MELI. Integrated enrichment analysis andpathway-based visualization of transcriptomic and metabolomic datawere conducted using IncroMap software 1.7 according to the KyotoEncyclopedia of Genes and Genomes (KEGG) pathway database, me-tabolism-related pathways with enrichment P values < 0.05 were se-lected and further sorted according to the pathway impact.
3. Results
3.1. SCCPs-induced changes in gene expression
Profiles of transcriptomic results for livers of male rats were ana-lyzed using cRNA microarrays. After removing unannotated and re-peated entities, a total of 10,144 genes were identified for which genesymbols were available. PLS-DA was performed to estimate differencesin expression of identified genes among groups, the R2 and Q2 were0.993 and 0.73 respectively. In the PLS-DA score plot, the first
component (PC1) distinguished the high-dose group from the otherdosed groups, and the middle-dose group was clearly separated fromthe control group by the second component (PC2). The low-dose groupand control group both had positive scores for PC1 and PC2 (Fig. 1A).Nevertheless, these groups were also distinct from each other. To verifythe results of the microarray analysis, eight differentially expressedgenes were selected randomly for validation and quantification by qRT-PCR. Overall, the qRT-PCR results closely paralleled patterns of ex-pression observed in the microarray data (Table S4).
Significantly greater values of TELI were observed with increaseddosages of SCCPs (Fig. 1B), which suggested that exposure to SCCPscaused perturbation of transcription, even at the low dose (0.01mg/kg bm/d). After removing mRNA with low signal intensity, pairwisecomparisons base on t-test were conducted to identify DEGs betweenthe control and rats exposed to SCCP. Genes displaying differences inexpression with a P value of < 0.05 and a fold change (FC) cut-offof > 1.5 were considered DEGs, after a 28-d exposure, 171, 213 and292 genes were identified to be DEGs in livers of rats exposed to 0.01, 1or 100mg/kg bm/d SCCPs, respectively (Fig. 1C).
A KEGG pathway enrichment analysis was then performed to de-termine the association of all DEGs with metabolic processes occurringin the rat livers, 63 DEGs were identified to be genes encoding hepaticenzymes (Table S5). Among these genes, 15, 20 and 42 were observedin rats exposed to 0.01, 1 or 100mg SCCPs/kg bm/d, respectively. Aheatmap of the hierarchical clustering analysis of DEGs encoding he-patic enzymes demonstrated three major dose-response trajectories(Fig. 1D). Expression levels of all 15 DEGs, encoding for enzymes in-volved in fatty acid metabolism were up-regulated in response to
Fig. 2. Changes in metabolic profile of SD rat liver after 28-day oral administration of SCCPs. (A) PLS-DA score plot of liver metabolites after exposed to SCCPs atvarious doses. (B) MELI of liver metabolism fingerprint in the control and SCCP-treated groups. (C) Metabolic correlation networks of the differential metabolites andrelated pathways. SM: sphingomyelin; PC: phosphatidyl choline; LysoPC: lysophosphatidyl choline; PE: phosphatidyl ethanolamine; LysoPE: lysophosphatidylethanolamine; PA: phosphatidic acid; CoA: coenzyme A; GSSH: oxidized glutathione; ADP: Adenosine diphosphate. (D) Hierarchical clustering based on the dif-ferential metabolites with P value of < 0.05 base on one-way ANOVA and VIP value of > 1, PEA:Palmitoylethanolamide; AEA: Anandamide; Leu-pro:Leucylproline; MG: monoacyl glycerol; DG: diacyl glycerols. *, P < 0.05; **, P < 0.01.
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exposure to SCCPs, whereas expression levels of four DEGs, encodingenzymes related to purine and pyrimidine metabolism, Enpp1, Enpp3,Prim1 and Pklr, were reduced in livers of rats exposed to SCCPs. Non-monotonic trends were observed for Elovl2, Elovl6, Fasn, Lpin1 andLpin2.
3.2. SCCP-induced changes in metabolic profiles
A PLS-DA was performed on all analyzed metabolites to estimate themetabolic variance induced by various doses of SCCPs. The R2 and Q2
were 0.979 and 0.69 respectively. In the PLS-DA score plot, rats ex-posed to the middle and high doses were clearly separated from thecontrol group, whereas rats exposed to the low dose exhibited a slightoverlap with the control group (Fig. 2A). Values of MELI exhibited asignificant and dose-dependent increase with SCCPs (Fig. 2B), whichsuggested that SCCP exposure perturbed normal metabolic pathwayseven at dose of 0.01mg/kg bm/d.
Among all doses, 328 metabolites exhibited statistically significant(one-way ANOVA) differences at P values < 0.05 and variable im-portance projection (VIP) value of > 1. Among these metabolites,based on pairwise comparisons with the control group, 127, 242 and328 metabolites were significantly (P < 0.05) different metabolites(DMs) in livers of rats exposed to 0.01, 1 and 100mg SCCPs/kg bm/d,respectively. The molecular structures of 118 DMs were reliably iden-tified by MS/MS, and some were further confirmed using authenticstandards (Table S6), a hierarchical clustering analysis of these identi-fied DMs between groups indicated two major dose-response trajec-tories (Fig. 2D). In trajectory I, concentrations of oxidized glutathione(GSSH), coenzyme A (CoA), adenosine diphosphate (ADP) and mostphospholipids were up-regulated in livers of rats exposed to all doses ofSCCPs. In trajectory II, concentrations of almost all fatty acids, aminoacids, purines, pyrimidines, intermediate metabolites in the tri-carboxylic acid (TCA) cycle and glucose were lower in livers of ratsexposed to SCCPs, relative to that in unexposed, control rats.
In a correlation network diagram constructed by pairwise compu-tation of Pearson correlations between DMs involved in the same KEGGmetabolic pathway (Fig. 2C). DMs were mainly connected by PCs, fattyacids and amino acids. Among differentially altered phospholipids,concentrations of PEs, LysoPEs and SMs were increased in response toSCCP exposure. However, PCs, LysoPCs and acylcarnitines exhibiteddifferent patterns. Concentrations of PCs linked to fatty acids weredecreased, whereas concentrations of PCs linked to PEs and LysoPEswere greater in rats exposed to SCCPs.
3.3. Integrated enrichment analysis of transcript and metabolite profiles
Integrated enrichment analysis indicate that exposure to SCCPsperturbed metabolism of linoleic acid, glycerophospholipid, sphingoli-pids and 2 pathways of amino acid (Cys and Met metabolism, Gly, Serand Thr metabolism), even at low dose of 0.01mg SCCPs/kg bm/d(Fig. 3). Nucleotide metabolism and unsaturated fatty acid biosynthesiswere also identified as the most relevant pathways affected by SCCPs atmiddle dose. carbohydrate metabolism, fatty acid degradation andperoxisome function were also significantly perturbed by SCCPs at highdose. Metabolic disorders in livers of rats fed 0.01 or 1mg/kg bmSCCPs/d were mainly characterized by changes in metabolome,whereas in livers of rats fed 100mg SCCPs/kg bm/d, the metabolicdisorder was caused by changes in transcriptome and metabolome.
3.4. Lipid metabolism alterations
Metabolomic and transcriptomic analyses demonstrated effects ofSCCPs on lipid metabolism. Interconnected pathways included glycer-ophospholipid metabolism, α-linoleic acid metabolism, sphingolipidsand arachidonic acid metabolism as well as elongation, biosynthesisand degradation of fatty acids (Fig. 4A). Concentrations of total fatty
acids (∑fatty acids) were inversely proportional to dose of SCCPs(Fig. 4B), whereas concentrations of total phospholipids (∑phospholi-pids) were directly proportional to dose of SCCPs (Fig. 4C). No sig-nificant difference was observed for total concentrations of acylcarni-tines (∑acylcarnitines; Fig. 4D). Specifically, the ratio of ω-6 fatty acidsto ω-3 fatty acids (ω-6/ω-3 ratio) was significantly greater in rats ex-posed to all doses of SCCPs, in a dose-dependent manner (Fig. 4E).
Degradation of fatty acids was greater in livers of rats exposed toSCCPs as ∑fatty acids in livers of rats fed 1 and 100mg SCCPs/kg bm/dwere significantly less by 29.5% and 42.3%, respectively. In livers ofrats dosed with 100mg SCCPs/kg bm/d, expression levels of severalgenes encoding enzymes involved in fatty acid β-oxidation and ω-oxi-dation were significantly greater than those of controls. Exposure toSCCPs also up-regulated the expression levels of the Elovl2 and Elovl6genes, which encode fatty acid elongases, Acot2, which encodes acyl-CoA thioesterase 2, and Fasn, which encodes a fatty acid synthase.Expression levels of two genes involved in arachidonic acid metabolism,Cyp2b12 and Cyp2j4, were up-regulated. Transformation of α-linoleicacid to stearidonic acid might be accelerated by up-regulation of Fads2.
SCCPs also disrupted metabolism of glycerophospholipid andsphingolipid. In glycerophospholipid metabolism, significantly higherconcentrations of phosphatidate were observed in livers of rats fed 1 or100mg SCCPs/kg bm/d than in controls. Expression levels of Lpin1 andLpin2, which encode phosphatidate phosphatases, were significantlyincreased in livers of rats dosed with 1 or 100mg SCCPs/kg bm/d.Moreover, transformations of PCs and PEs to LysoPCs and LysoPEs weresignificantly altered (Fig. 4A and Fig. S3), which might be due to down-regulated expression of Pla2g16, which encodes phospholipase A2. Insphingolipid metabolism, concentrations of SMs were greater in all ratsexposed to SCCPs and expression levels of Acer2 and Asah2, whichencode alkaline ceramidase 2 and neutral ceramidase, respectively,were significantly up-regulated in livers of rats fed 1 or 100mg/kg bm/d, respectively. In addition, expression of Neu2, which encodes neur-aminidase 2, was also up-regulated in livers of rats exposed to1mg SCCPs/kg bm/d.
3.5. Amino acid metabolism alterations
Results of metabolomic analyses indicated depletion of amino acidsin livers of rats exposed to SCCPs. Concentrations of total amino acids(∑amino acids) in livers of rats exposed to SCCPs, were significantlydecreased by more than 20% (Fig. 5A). The most relevant pathways foramino acid metabolism altered by exposure to SCCPs were alanine,
Fig. 3. Most relevant metabolic pathways perturbed by exposure to SCCPs atvarious doses based on the integrated enrichment analysis of transcript andmetabolites.
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aspartate and glutamate metabolism; glycine, serine and threoninemetabolism (Fig. 5B). However, results of transcriptomic analysesidentified only one DEG that directly regulates amino acid metabolism,expression of Alas1, which encodes 5′-aminolevulinate synthase 1, wassignificantly up-regulated in livers of rats exposed to 0.01 and 100mg/kg bm/d groups.
During catalysis by glutathione (GSH) synthase, glycine is trans-formed to GSH by linking it to L-γ-glutamylcysteine. Although con-centrations of glycine and GSH in rat livers were not significantly al-tered by SCCP exposure, concentrations of GSSH were significantlyincreased in all three SCCP-treated groups. Moreover, expression ofGpx3, which encodes glutathione peroxidase 3 that catalyzes thetransformation of GSH to GSSH, was significantly increased in ratsexposed to 1mg SCCPs/kg bm/d (Fig. 5B). In addition, by increasingthe glutathione S-transferase capacity for the purpose of detoxification,exposure to SCCPs accelerated conjugation of GSH to SCCP to formSCCP-S-glutathione (Buryskova et al., 2006). Expression of Gstm5,which encodes the enzyme glutathione S-transferase mu 5 that cata-lyzes conjugation of GSH to organic halides (RXs) to form R-S-glu-tathione, was significantly increased in livers of rats exposed to100mg SCCPs/kg bm/d, relative to that in livers of rats in the controlgroup.
3.6. Carbohydrate metabolism alterations
Exposure to SCCPs resulted in decreased glycolysis/
gluconeogenesis. Concentrations of glucose were significantly de-creased in livers of rats exposed to 100mg SCCPs/kg bm/d, and con-centrations of L-lactate were decreased in livers of rats exposed to 1 or100mg SCCPs/kg bm/d. In addition, expressions of Fbp2 and Pklr,which encodes fructose-bisphosphatase 2 and pyruvate kinase, weresignificantly decreased in livers of rats exposed to 100mg SCCPs/kg bm/d.
Regarding the TCA cycle, concentrations of succinate and 2-ox-oglutarate exhibited a decreasing trend in rats exposed to SCCPs, andconcentrations of malate were less in livers of rats exposed to 1 or100mg SCCPs/kg bm/d groups, which suggested that SCCPs suppressedthe central hub of oxidative metabolism. However, pyruvate metabo-lism and propanoate metabolism might be enhanced by exposure toSCCPs, mainly via up-regulation expression of the Me1 gene, whichencodes malate dehydrogenase, the Ehhadh gene, which encodes enoyl-CoA hydratase, and the Hibch gene, which encodes 3-hydro-xyisobutyryl-CoA hydrolase. Among these three genes, Me1 and Ehhadhare both target genes of PPARα (Burri et al., 2010; Ehara et al., 2015).In addition, the levels of CoA were significantly increased in livers ofrats exposed to middle and high dose of SCCPs, which should acceleratethe synthesis and oxidation of fatty acids. All of these effects wouldresult in overall suppression of the TCA cycle. The elevated con-centrations of CoA might result from induction of the expression of thePpcdc gene, which encodes phosphopantothenoylcysteine decarbox-ylase (Fig. 5B).
Fig. 4. Disorder of lipid metabolism in liver of SD rat after 28-day oral administration of SCCPs. (A) Lipid metabolism featuring the most relevant metabolites andgenes perturbed by SCCPs. Statistically significant changes in metabolites and gene expression are shown in the boxes as red (up-regulation) or green (down-regulation). Genes were shown in italic to distinguish with metabolites. (B), (C) and (D) variation tendency of total fatty acids, total phospholipids and totalacylcarnitines in liver tissue from control and SCCPs exposure rats. (E) Polyunsaturated fatty acids ω-6/ω-3 ratio in liver tissue of rats increased for three SCCPsexposure groups. *, P < 0.05; **, P < 0.01.
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3.7. Nucleotide metabolism alterations
Total concentrations of pyrimidines (∑pyrimidines) were sig-nificantly decreased in livers of rats exposed to SCCPs, and total con-centrations of purines (∑purines) were significantly decreased in 1 or100mg SCCPs/kg bm/d (Fig. 6A and B), which suggests that exposureto SCCPs disrupts nucleotide metabolism. In pyrimidine metabolism,concentrations of cytosine and thymine were both significantly reducedby more than 30% in livers of rats exposed to SCCP, compared to thecontrols. In purine metabolism, concentrations of guanine, xanthosine,xanthine, uric acid, and deoxyinosine were all 50% less in livers of ratsexposed to 100mg SCCPs/kg bm/d. However, results of the tran-scriptomic analysis did not identify any DEGs that directly regulatetransformation of pyrimidines or purines (Fig. 6C). Only two DEGs thatregulate replication and repair of DNA were observed in livers of ratsexposed to 100mg SCCPs/kg bm/d. Expression of Prim1, which encodesDNA primase subunit 1, was significantly decreased, whereas expres-sion of Pold2, which encodes DNA polymerase delta subunit 2, wassignificantly greater in rats exposed to SCCPs.
Exposure to SCCPs inhibited ATP turnover (Fig. 6C). Decreasedexpression of Enpp1 and Enpp3 genes, both of which encode ectonu-cleotide pyrophosphatases, suggests that transformation of ATP toadenosine monophosphate (AMP) or ADP was inhibited by SCCPs. In-creased concentrations of ADP and down-regulated expression of theAtp5hl1 gene, encoding ATP synthase, indicated that conversion of ADPto ATP was also inhibited by SCCPs. Moreover, the expression of thePklr encoding pyruvate kinase which can catalyze conversion of ADP toATP was also significantly down-regulated in livers of rats exposed to100mg SCCPs/kg bm/d. In addition, expression of Adcy10, which en-codes adenylate cyclase 10, was significantly increased in livers of ratsexposed to 0.01 or 100mg SCCPs/kg bm/d groups. Inhibition of theturnover of ATP-ADP-AMP would inevitably limit energy production.
4. Discussion
Due to the function of PPARα on lipid metabolism and peroxisomeproliferation, and the structural similarity between SCCPs and naturalligands (fatty acids) for PPARα (Krey et al., 1997), it was speculatedthat SCCPs might activate PPARα and lead to adverse effects on me-tabolism in hepatocytes, however, no direct evidence of activation ofPPARα by SCCPs has been reported previously. In the present study,this hypothesis was verified by use of a combination of metabolomicsand transcriptomics and the results suggest two major modes of toxicaction for SCCPs: (1) inhibition of energy metabolism and (2) activationof PPARα, and demonstrated possible effects of health due to chronicexposure to low-dose of SCCPs.
4.1. Evidences of SCCP-induced activation of PPARa
Results of this study suggest that SCCPs caused hepatotoxicity byactivation of PPARα. PPARα is a transcription factor and a major reg-ulator of hepatic lipid metabolism. Upon activation, PPARα forms aheterodimer with the retinoid X receptor (RXR) and then binds to aspecific peroxisome proliferator response element (PPRE) in or aroundtarget genes. 16 genes regulated by PPARα (Golla et al., 2016;Rakhshandehroo et al., 2010; Zhang et al., 2018), including Hadhb,Eci1, Decr2, Ech1, Ehhadh, Acaa1a, Acaa1b, Acot2, Fasn, Fads2, Cyp4a1,Elovl2, Elovl6, Lpin1, Lpin2 and Me1, were up-regulated after exposureto SCCPs (Figs. 4A and 5B), indicating that exposure to SCCPs activatedthe PPARα signaling pathway. Among them, 13 genes were involved infatty acid metabolism, which suggests that oxidation, biosynthesis andelongation of fatty acids were stimulated. In addition, decreased ∑fattyacids in livers of rats exposed to SCCPs further indicated the inductionof fatty acid metabolism. In several toxicological studies of SCCPs onmale rats, increased proliferation of peroxisomes, hepatocellular
Fig. 5. Disorder of amino acid and carbohydrate metabolism in liver of SD rat after 28-day oral administration of SCCPs. (A) Total concentrations of amino acids(∑Amino acids) in liver of SD rat of the control group and three doses of SCCPs. (B) Most relevant amino acids perturbed by SCCPs on the basis of the pathwayenrichment analysis. GSH: glutathione, GSSH: oxidized glutathione, **, P < 0.01.
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carcinoma and greater liver weight were observed, all of which areconsistent with activation of PPARα (Bucher et al., 1987; Nilsen et al.,1981; Wyatt et al., 1993). In the present study, greater liver weight wasalso observed at dose of SCCPs near the LOAEL (Fig. S2C). In addition,five genes (Fbp2, Acot, Decr2, Hadhb, Hibch) were up-regulated by a foldchange of> 1.5 in mouse liver after exposure to three PPARα activa-tors (Oshida et al., 2015), which verified the PPARα activation effect ofSCCPs. Moreover, agonism of SCCPs towards the rat PPARα was con-firmed by luciferase reporter gene assays. Exposure to SCCPs caused adose-dependent increase in luciferase activity in rat PPARα-transfectedkidney 293T cells, which was a more than 20% increase for the luci-ferase activity when exposed to doses of SCCPs > 1 µmol/L (Gonget al., 2019). These results demonstrated the transactivation potency ofSCCPs towards rat PPARα.
4.2. SCCPs inhibit energy metabolism
Energy metabolism is the process by which ATP is generated viaoxidative phosphorylation and glycolysis. In the present study, ex-posure to SCCPs inhibited energy production from oxidative phos-phorylation, as determined by an increase in concentrations of ADP andlesser expression of Atp5hl1. Oxidative phosphorylation comprises re-spiratory chain complexes I to IV together with ATP synthase (complexV). Lesser expression of Atp5hl1 might result in a deficit in ATP syn-thase. Moreover, exposure to SCCPs affected transfer of electrons in the
respiratory chain. SCCPs blocked transfer of electrons in respiratorycomplex II (succinate dehydrogenase). This enzyme complex oxidizessuccinate to fumarate and transfers electrons through three iron-sulfurclusters to ubiquinone, and thus participates in both the TCA cycle andthe electron transport chain. In this study, concentrations of succinate,2-oxoglutarate and malate were less in livers of rats exposed to SCCPs,compared to that in the control group (Fig. 5B), which indicated in-hibition of the TCA cycle. The decreased levels of succinate and in-hibition of the TCA cycle would inevitably decrease efficiency of elec-tron transfer to ubiquinone. Alternatively, exposure to SCCPs mightstimulate transfer of electrons in respiratory complex I (NADH: ubi-quinone oxidoreductase) by accelerating oxidation of fatty acidsthrough activation of the PPARα.
In rat hepatocytes, oxidative phosphorylation occurs in mitochon-dria. SCCPs inhibited oxidative phosphorylation implies that the mi-tochondrion is an important target of SCCPs. Mitochondrial dysfunctionis likely a major mechanism of SCCPs induced liver toxicity (Porcedduet al., 2012). It was speculated that oxidative phosphorylation wassuppressed partially to minimize ROS production. In the present study,elevation of GSSH in livers of rats exposed to all doses of SCCPs suggeststhat ROS-induced oxidative stress was significantly increased (Fig. 5B).As evidenced by up-regulation of Pold2, which encodes DNA poly-merase delta subunit 2 (Fig. 6B), SCCPs may cause damage to DNA, andsuppression of oxidative phosphorylation is a biochemical response toSCCP-induced DNA damage (Zhang et al., 2017).
Fig. 6. Disorder of nucleotide metabolism in liver of SD rat after 28-day oral administration of SCCPs. (A) and (B) Total concentrations of pyrimidines (∑Pyrimidines)and purines (∑Purines) in liver of unexposed (control) SD rat or three oral doses of SCCPs. (C) Most relevant metabolites and differentially expressed genes perturbedby SCCPs in nucleotide metabolism, **, P < 0.01.
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Exposure to SCCPs inhibited glycolysis, which is another pathwayfor generation of ATP. Down-regulation of Pklr exposure to high doseSCCPs might decrease activity of pyruvate kinase and thus decreasegeneration of ATP via glycolysis (Fig. 5B). Because pyruvate kinasecatalyzes the transphosphorylation of phohsphoenolpyruvate into pyr-uvate and ATP, which is the rate-limiting step of glycolysis. As a directdegradation product of pyruvate, concentration of L-lactate also ex-hibited a decreasing trend in livers of rats in two SCCP-treated groupscompared with the control group. Also, activity of lactate dehy-drogenase was also significantly decreased in blood of rats exposed to1mg SCCPs/kg bm/d (Fig. S2D), which further verified that SCCPsinhibited glycolysis. Lesser concentrations of glucose in livers of ratsexposed to SCCPs might have resulted from suppression of gluconeo-genesis, which is a major contributor to blood glucose homeostasis(Wang et al., 2018a). In the gluconeogenesis pathway, expression ofFbp2 was significantly decreased in livers of rats exposed to100mg SCCPs/kg bm/d, compared with expression in the control rats(Fig. 5B). The enzyme encode by Fbp2 can catalyze the conversion offructose-1,6-bisphosphate to fructose 6-phosphate, which is the rate-limiting step in gluconeogenesis.
Inhibition of ATP-ADP-AMP turnover by SCCPs was characterizedby the decreased expression levels of Atp5hl1, pklr, Enpp1 and Enpp3(Fig. 6C). Inhibited conversion of ATP to AMP and reduced con-centrations of blood glucose might stimulate the activity of adenylatecyclase to maintain or increase concentrations of cAMP, which is a keysecond messenger molecule affecting multiple cellular processes in theliver. High concentrations of cAMP have been reported to increaseglucose production by stimulating gluconeogenesis and glycogenolysis(Huang et al., 2013; Wahlang et al., 2018). In the present study, up-regulation of Adcy10 was observed in livers of rats exposed to SCCPs(Fig. 6C). Inhibition of oxidative phosphorylation, glucose metabolismand ATP turnover, together with greater concentrations of ADP, suggestthat exposure to SCCPs substantially inhibited energy metabolism inlivers of rat. Consequently, the active transport of nutrients such asamino acids, glucose and nucleotides would be inevitably reduced. Inthe study, results of which are presented here, the concentrations of∑amino acids, ∑pyrimidines and ∑purines were all remarkably reducedin the livers of rats exposed to the three doses of SCCPs (Figs. 5A, 6Aand 6B). In an in vitro study, significant reductions in Na+/K+-ATPaseactivity and concentrations of ATP were observed in HepG2 cells ex-posed to SCCPs (Wang et al., 2018b).
4.3. Possible health effects of exposure to low-dose SCCPs
With their large and widespread use in industrial applications,SCCPs have been ubiquitously detected in environment and the humanbody at low doses. In the present study, it was found that SCCPs causedsignificant alterations in metabolism and energy conversion in the liverof SD rats even at environmentally relevant doses. Inhibition of energymetabolism resulted in alterations in active transport of amino acids,glucose and nucleotides and thus resulted in lesser concentrations ofthreonine, serine, methionine, asparagine, glutamine, thymine and cy-tosine in livers of rats exposed to all three doses of SCCPs (Figs. 5B and6B), that might inhibit a series of biological reactions, including aminoacid metabolism, nucleotide metabolism, protein biosynthesis, DNAreplication and DNA repair, all of which would ultimately affectgrowth. Lower body mass gain has been observed in rats exposed toSCCPs at environmentally relevant doses. Moreover, pathophysiologicalanalysis also indicated that exposure to SCCPs at environmentally re-levant doses induced a considerable reduction in creatine kinase ac-tivity, which might results from inhibition of energy metabolism. Be-cause livers of rats and human share similar mechanisms and pathwaysfor energy metabolism, it was speculated that exposure to SCCPs atenvironmentally relevant doses would inhibit energy metabolism inhuman liver. In previous in vitro studies, it was observed that exposureto SCCPs at environmentally relevant doses resulted in lower
concentrations of amino acids and nucleotides in human HepG2 hepa-tocytes (Geng et al., 2015; Wang et al., 2018b). Inhibition of energymetabolism in the human liver might cause nutrient deficiency and lossof body mass.
Although activation of PPARα by SCCPs was observed at high dosein this study, an acceleration of fatty acid metabolism, which mainlyresulted from the SCCP-induced activation of a small amount of PPARα,was caused by exposure to SCCPs at environmentally relevant doses.Rats and humans share similar tissue distribution profiles for expressionof PPARα (Mukherjee et al., 1994). While, because expression of mRNAfor PPARα was only at low levels in human liver and humans are lesssensitive than rodents to effect of peroxisome proliferation (Bility et al.,2004; Palmer et al., 1998), the importance of PPARα in human liver hasbeen questioned. However, results of recent studies using more ad-vanced methodologies revealed similar PPARα levels in some people tothose observed in mice and rats (Walgren et al., 2000), which providedevidence against this hypothesis. Therefore, SCCP-induced hepatotoxi-city that occurs via interactions with PPARα should be carefully eval-uated in vulnerable subpopulations of humans.
The balance of ω-6 and ω-3 fatty acids in an organism is essential fornormal growth and development, and the ω-6/ω-3 ratio plays an im-portant role in tumorigenesis (Xia et al., 2006, 2005). The dose-de-pendent increase in ω-6/ω-3 ratio suggests that rats exposed to SCCPshad a high risk of developing tumors and cancer. Results of a 2-yearstudy in which rats were exposed to SCCPs indicate the potential car-cinogenicity (Bucher et al., 1987), and SCCPs have been classified, in1990, by the International Agency for Research on Cancer (IARC) as aGroup 2B carcinogen, that is possibly carcinogenic to humans (IARC,1990).
5. Conclusions
Exposure of rats to SCCPs was shown to cause hepatotoxicity atenvironmentally relevant dosages. Changes to metabolism of lipids,amino acids and nucleotides in livers of rats after low-dose exposure toSCCPs were linked to transcriptomic changes, and might partly explainthe results of histological examinations, blood glucose and body masschanges in rat exposed to SCCPs at the high dose. SCCPs might act asPPARα agonists and up-regulating relevant expression of genes, sti-mulating β-oxidation of unsaturated fatty acids (Geng et al., 2015) aswell as peroxisome proliferation in liver of rodents (Wyatt et al., 1993).Exposure to SCCPs suppresses oxidative phosphorylation, glycolysis,gluconeogenesis and ATP-ADP-AMP turnover, and thus induces aminoacid and nucleotide deficiencies in livers of rat. Significant SCCP-in-duced inhibition of energy metabolism occurs at environmentally re-levant doses, which suggests that SCCPs exhibit significant effects onmetabolism. Collectively, results of the current study provide under-standing for the molecular mechanisms of hepatotoxicity and adversehealth effects caused by exposure to SCCPs.
Limitations and future directionsIn this study, omic techniques were found to be powerful tools to
observe global biological changes following exposures to SCCPs, even atlow doses. Results of this study raise importance regarding the role ofPPARα and energy metabolism to SCCPs induced hepatotoxicity in SDrat. However, this study has some limitations. Thus, to further verifyagonism of SCCPs to the PPARα, expression/knockdown experimentsshould be performed to make this conclusion more convincing. Toverify the inhibition of energy metabolism caused by SCCPs, a detailedevaluation of mitochondrial respiration and glycolysis, the two majorenergy-producing pathways in the cell, will be performed. In particular,in view of the fact that rat and human livers share similar mechanismsfor energy metabolism and similar PPARα tissue expression profiles,work is ongoing based on a human hepatic cell-line to explore thepossible health effects induced by SCCPs. In our future studies, systemverification will be performed to provide a better understanding for themechanisms of SCCPs induced liver toxicity.
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Declaration of Competing Interest
The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influ-ence the work reported in this paper.
Acknowledgments
This work was financially supported by National Basic ResearchProgram of China (grant no. 2015CB453100) and National NaturalScience Foundation of China (grant nos. 21607152, 21277141,21337002 and 91543201).
Appendix A. Supplementary material
Gene expression analysis; LC-MS-based metabonomic analysis;Determination of TELI and MELI; General pathophysiological results;Detail information of 63 DEGs and 118 DMs; Results of pathophysio-logical tests and RT-PCR validation were offered in SupplementalMaterial. Supplementary data to this article can be found online athttps://doi.org/10.1016/j.envint.2019.105231.
References
Ali, T.E.-S., Legler, J., 2010. Overview of the mammalian and environmental toxicity ofchlorinated paraffins. In: Boer, J. (Ed.), Chlorinated Paraffins. Springer, Berlin,Heidelberg, pp. 135–154.
Bility, M.T., Thompson, J.T., McKee, R.H., David, R.M., Butala, J.H., Vanden Heuvel, J.P.,Peters, J.M., 2004. Activation of mouse and human peroxisome proliferator-activatedreceptors (PPARs) by phthalate monoesters. Toxicol. Sci. 82, 170–182. https://doi.org/10.1093/toxsci/kfh253.
Bucher, J.R., Alison, R.H., Montgomery, C.A., Huff, J., Haseman, J.K., Farnell, D.,Thompson, R., Prejean, J.D., 1987. Comparative toxicity and carcinogenicity of twochlorinated paraffins in F344/N rats and B6C3F1 mice. Fundam. Appl. Toxicol. 9,454–468. https://doi.org/10.1093/toxsci/9.3.454.
Burri, L., Thoresen, G.H., Berge, R.K., 2010. The role of PPAR activation in liver andmuscle. PPAR Res. 2010, 542359. https://doi.org/10.1155/2010/542359.
Buryskova, B., Blaha, L., Vrskova, D., Simkova, K., Marsalek, B., 2006. Sublethal toxiceffects and induction of glutathione S-transferase by short chain chlorinated paraffins(SCCPs) and C-12 alkane (dodecane) in Xenopus laevis frog embryos. Acta Vet. Brno75, 115–122. https://doi.org/10.2754/avb200675010115.
International Agency for Research on Cancer (IARC), 1990. Some Flame Retardants andTextile Chemicals, and Exposures in the Textile Manufacturing Industry. IARCMonographs on the Evaluation of Carcinogenic Risks To Humans, Lyon, France, pp.55–72.
Cao, Y., Harada, K.H., Liu, W.Y., Yan, J.X., Zhao, C., Niisoe, T., Adachi, A., Fujii, Y.,Nouda, C., Takasuga, T., Koizumi, A., 2015. Short-chain chlorinated paraffins incooking oil and related products from China. Chemosphere 138, 104–111. https://doi.org/10.1016/j.chemosphere.2015.05.063.
Cao, Y., Harada, K.H., Hitomi, T., Niisoe, T., Wang, P., Shi, Y., Yang, H.R., Takasuga, T.,Koizumi, A., 2017. Lactational exposure to short-chain chlorinated paraffins in China,Korea, and Japan. Chemosphere 173, 43–48. https://doi.org/10.1016/j.chemosphere.2016.12.078.
Chen, H., Lam, J.C.W., Zhu, M.S., Wang, F., Zhou, W., Du, B.B., Zeng, L.X., Zeng, E.Y.,2018. Combined effects of dust and dietary exposure of occupational workers andlocal residents to short- and medium-chain chlorinated paraffins in a mega e-wasterecycling industrial park in south China. Environ. Sci. Technol. 52, 11510–11519.https://doi.org/10.1021/acs.est.8b02625.
Ehara, T., Kamei, Y., Yuan, X., Takahashi, M., Kanai, S., Tamura, E., Tsujimoto, K.,Tamiya, T., Nakagawa, Y., Shimano, H., Takai-Igarashi, T., Hatada, I., Suganami, T.,Hashimoto, K., Ogawa, Y., 2015. Ligand-activated PPARα-dependent DNA de-methylation regulates the fatty acid β-oxidation genes in the postnatal liver. Diabetes64, 775–784. https://doi.org/10.2337/db14-0158.
Fiedler, H., 2010. Short-chain chlorinated paraffins: production, use and internationalregulations. In: Boer, J. (Ed.), Chlorinated Paraffins. Springer, Berlin, Heidelberg, pp.1–40.
Friden, U.E., McLachlan, M.S., Berger, U., 2011. Chlorinated paraffins in indoor air anddust: concentrations, congener patterns, and human exposure. Environ. Int. 37,1169–1174. https://doi.org/10.1016/j.envint.2011.04.002.
Gao, W., Cao, D.D., Wang, Y.J., Wu, J., Wang, Y., Wang, Y.W., Jiang, G.B., 2017. Externalexposure to short- and medium-chain chlorinated paraffins for the general populationin Beijing, China. Environ. Sci. Technol. 52, 32–39. https://doi.org/10.1021/acs.est.7b04657.
Gao, Y., Zhang, H.J., Chen, J.P., Zhang, Q., Tian, Y.Z., Qi, P.P., Yu, Y.Z., 2011. Optimizedcleanup method for the determination of short chain polychlorinated n-alkanes insediments by high resolution gas chromatography/electron capture negative ion–lowresolution mass spectrometry. Anal. Chim. Acta 703, 187–193. https://doi.org/10.1016/j.aca.2011.07.041.
Geng, N.B., Zhang, H.J., Zhang, B.Q., Wu, P., Wang, F.D., Yu, Z.K., Chen, J.P., 2015.Effects of short-chain chlorinated paraffins exposure on the viability and metabolismof human hepatoma HepG2 cells. Environ. Sci. Technol. 49, 3076–3083. https://doi.org/10.1021/es505802x.
Gluge, J., Wang, Z.Y., Bogdal, C., Scheringer, M., Hungerbuhler, K., 2016. Global pro-duction, use, and emission volumes of short-chain chlorinated paraffins - a minimumscenario. Sci. Total Environ. 573, 1132–1146. https://doi.org/10.1016/j.scitotenv.2016.08.105.
Golla, U., Joseph, D., Tomar, R.S., 2016. Combined transcriptomics and chemical-geneticsreveal molecular mode of action of valproic acid, an anticancer molecule usingbudding yeast model. Sci. Rep. 6, 35322. https://doi.org/10.1038/srep35322.
Gong, Y.F., Zhang, H.J., Geng, N.B., Xing, L.G., Fan, J.F., Luo, Y., Song, X.Y., Ren, X.Q.,Wang, F.D., Chen, J.P., 2018. Short-chain chlorinated paraffins (SCCPs) inducedthyroid disruption by enhancement of hepatic thyroid hormone influx and de-gradation in male Sprague Dawley rats. Sci. Total Environ. 625, 657–666. https://doi.org/10.1016/j.scitotenv.2017.12.251.
Gong, Y.F., Zhang, H.J., Geng, N.B., Ren, X.Q., Giesy, J.P., Luo, Y., Xing, L.G., Wu, P., Yu,Z.K., Chen, J.P., 2019. Short-chain chlorinated paraffins (SCCPs) disrupt hepatic fattyacid metabolism in liver of male rat via interacting with peroxisome proliferator-activated receptor α (PPARα). Ecotox. Environ. Safe 2019 (181), 164–171. https://doi.org/10.1016/j.ecoenv.2019.06.003.
Hüttig, J., Oehme, M., 2005. Presence of chlorinated paraffins in sediments from theNorth and Baltic Seas. Arch. Environ. Contam. Toxicol. 49, 449–456. https://doi.org/10.1007/s00244-005-7024-7.
Krey, G., Braissant, O., L’Horset, F., Kalkhoven, E., Perroud, M., Parker, M.G., Wahli, W.,1997. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands ofperoxisome proliferator-activated receptors by coactivator-dependent receptor ligandassay. Mol. Endocrinol. 11, 779–791. https://doi.org/10.1210/mend.11.6.0007.
Li, H.J., Fu, J.J., Zhang, A.Q., Zhang, Q.H., Wang, Y.W., 2016. Occurrence, bioaccumu-lation and long-range transport of short-chain chlorinated paraffins on the FildesPeninsula at King George Island. Antarctica. Environ. Int. 94, 408–414. https://doi.org/10.1016/j.envint.2016.05.005.
Li, T., Wan, Y., Gao, S.X., Wang, B.L., Hu, J.Y., 2017. High-throughput determination andcharacterization of short-, medium-, and long-chain chlorinated paraffins in humanblood. Environ. Sci. Technol. 51, 3346–3354. https://doi.org/10.1021/acs.est.6b05149.
Liu, L.H., Li, Y.F., Coelhan, M., Chan, H.M., Ma, W.L., Liu, L.Y., 2016. Relative devel-opmental toxicity of short-chain chlorinated paraffins in Zebrafish (Danio rerio)embryos. Environ. Pollut. 219, 1122–1130. https://doi.org/10.1016/j.envpol.2016.09.016.
Mukherjee, R., Jow, L., Noonan, D., McDonnell, D.P., 1994. Human and rat peroxisomeproliferator activated receptors (PPARs) demonstrate similar tissue distribution butdifferent responsiveness to PPAR activators. J. Steroid Biochem. 51, 157–166.https://doi.org/10.1016/0960-0760(94)90089-2.
Nielsen, E., Ladefoged, O. (Eds.), 2013. Evaluation of Health hazards by Exposure toChlorinated Paraffins and Proposal of A Health-Based Quality Criterion for AmbientAir. The Danish Environmental Protection Agency, Copenhagen.
Nilsen, O.G., Toftgard, R., Glaumann, H., 1981. Effects of chlorinated paraffins on ratliver microsomal activities and morphology. Arch. Toxicol. 49, 1–13. https://doi.org/10.1007/BF00352066.
Oshida, K., Vasani, N., Thomas, R.S., Applegate, D., Rosen, M., Abbott, B., Lau, C., Guo,G., Aleksunes, L.M., Klaassen, C., Corton, J.C., 2015. Identification of modulators ofthe nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) ina mouse liver gene expression compendium. PLoS ONE 10, e0112655. https://doi.org/10.1371/journal.pone.0112655.
Porceddu, M., Buron, N., Roussel, C., Labbe, G., Fromenty, B., Borgne-Sanchez, A., 2012.Prediction of liver injury induced by chemicals in human with a multiparametricassay on isolated mouse liver mitochondria. Toxicol. Sci. 129, 332–345. https://doi.org/10.1093/toxsci/KFS197.
Rakhshandehroo, M., Knoch, B., Muller, M., Kersten, S., 2010. Peroxisome proliferator-activated receptor alpha target genes. PPAR Res. 2010, 612089. https://doi.org/10.1155/2010/612089.
Shi, L.M., Gao, Y., Zhang, H.J., Geng, N.B., Xu, J.Z., Zhan, F.Q., Ni, Y.W., Hou, X.H., Chen,J.P., 2017. Concentrations of short- and medium-chain chlorinated paraffins in in-door dusts from malls in china: Implications for human exposure. Chemosphere 172,103–110. https://doi.org/10.1016/j.chemosphere.2016.12.150.
Shi, L.M., Reid, L.H., Jones, W.D., Shippy, R., Warrington, J.A., Baker, S.C., et al., 2006.The microarray quality control (maqc) project shows inter- and intraplatform re-producibility of gene expression measurements. Nat. Biotechnol. 24, 1151–1161.https://doi.org/10.1038/nbt1239.
Spivey, A., 2004. Systems biology: the big picture. Environ. Health Perspect. 112,A938–A943. https://doi.org/10.1289/ehp.112-a938.
Thomas, G.O., Farrar, D., Braekevelt, E., Stern, G., Kalantzi, O.I., Martin, F.L., Jones, K.,2006. Short and medium chain length chlorinated paraffins in UK human milk fat.Environ. Int. 32, 34–40. https://doi.org/10.1016/j.envint.2005.04.006.
Hormones and endocrine-disrupting chemicals: Low-dose effects and nonmonotonicdose responses. Endocr. Rev. 33, 378–455. https://doi.org/10.1210/er.2011-1050.
Wahlang, B., McClain, C., Barve, S., Gobejishvili, L., 2018. Role of cAMP and phospho-diesterase signaling in liver health and disease. Cell. Signal. 49, 105–115. https://doi.org/10.1016/j.cellsig.2018.06.005.
Walgren, J.E., Kurtz, D.T., McMillan, J.M., 2000. Expression of PPAR(alpha) in humanhepatocytes and activation by trichloroacetate and dichloroacetate. Res. Commun.Mol. Pathol. Pharmacol. 108, 116–132.
Wang, B., Smyl, C., Chen, C.-Y., Li, X.-Y., Huang, W., Zhang, H.-M., Pai, V.J., Kang, J.X.,2018a. Suppression of postprandial blood glucose fluctuations by a low-carbohy-drate, high-protein, and high-omega-3 diet via inhibition of gluconeogenesis. Int. J.Mol. Sci. 19, 1823. https://doi.org/10.3390/ijms19071823.
Wang, F.D., Zhang, H.J., Geng, N.B., Ren, X.Q., Zhang, B.Q., Gong, Y.F., Chen, J.P.,2018b. A metabolomics strategy to assess the combined toxicity of polycyclic aro-matic hydrocarbons (PAHs) and short-chain chlorinated paraffins (SCCPs). Environ.Pollut. 234, 572–580. https://doi.org/10.1016/j.envpol.2017.11.073.
Wei, G.L., Liang, X.L., Li, D.Q., Zhuo, M.N., Zhang, S.Y., Huang, Q.X., Liao, Y.S., Xie, Z.Y.,Guo, T.L., Yuan, Z.J., 2016. Occurrence, fate and ecological risk of chlorinated par-affins in Asia: a review. Environ. Int. 92–93, 373–387. https://doi.org/10.1016/j.envint.2016.04.002.
Wyatt, I., Coutss, C.T., Elcombe, C.R., 1993. The effect of chlorinated paraffins on hepaticenzymes and thyroid hormones. Toxicology 77, 81–90. https://doi.org/10.1016/0300-483X(93)90139-J.
Xia, D., Gao, L.R., Zheng, M.H., Li, J.G., Zhang, L., Wu, Y.N., Tian, Q.C., Huang, H.T.,Qiao, L., 2017. Human exposure to short- and medium-chain chlorinated paraffins viamothers' milk in chinese urban population. Environ. Sci. Technol. 51, 608–615.https://doi.org/10.1021/acs.est.6b04246.
Xia, S.H., Lu, Y., Wang, J.D., He, C.W., Hong, S., Serhan, C.N., Kang, J.X., 2006.Melanoma growth is reduced in fat-1 transgenic mice: Impact of omega-6/omega-3essential fatty acids. Proc. Natl. Acad. Sci. USA 103, 12499–12504. https://doi.org/10.1073/pnas.0605394103.
Xia, S.H., Wang, J.D., Kang, J.X., 2005. Decreased n-6/n-3 fatty acid ratio reduces theinvasive potential of human lung cancer cells by downregulation of cell adhesion/invasion-related genes. Carcinogenesis 26, 779–784. https://doi.org/10.1093/carcin/bgi019.
Zhang, C., Skamagki, M., Liu, Z., Ananthanarayanan, A., Zhao, R., Li, H., Kim, K., 2017.Biological significance of the suppression of oxidative phosphorylation in inducedpluripotent stem cells. Cell Rep. 21, 2058–2065. https://doi.org/10.1016/j.celrep.2017.10.098.
Zhang, Q., Wang, J.H., Zhu, J.Q., Liu, J., Zhang, J.Y., Zhao, M.R., 2016. Assessment of theendocrine-disrupting effects of short-chain chlorinated paraffins in in vitro models.Environ. Int. 94, 43–50. https://doi.org/10.1016/j.envint.2016.05.007.
Zhang, W.L., Zhong, W., Sun, Q., Sun, X.G., Zhou, Z.X., 2018. Adipose-specific lipin1overexpression in mice protects against alcohol-induced liver injury. Sci. Rep. 8, 408.https://doi.org/10.1038/s41598-017-18837-2.
N. Geng, et al. Environment International 133 (2019) 105231
Purine metabolism Pyrimidine metabolism Starch and sucrose metabolism Riboflavin metabolism Nicotinate and nicotinamide metabolism Pantothenate and CoA biosynthesis
Purine metabolism Pyrimidine metabolism Starch and sucrose metabolism Riboflavin metabolism Nicotinate and nicotinamide metabolism Pantothenate and CoA biosynthesis