Promoting differentiation and lipid metabolism are the primary effects for DINP exposure on 3T3-L1 preadipocytes

Environmental Pollution 255 (2019) 113154

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Promoting differentiation and lipid metabolism are the primary effects for DINP exposure on 3T3-L1 preadipocytes* Lianying Zhang a, b, Weijie Sun a, Xiaoyu Duan a, Yishuang Duan a, Hongwen Sun a, * a

Ministry of Education Key Laboratory of Pollution Processes and Environmental Criteria, College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China b School of Environmental Science and Safety Engineering, Tianjin University of Technology, Tianjin 300384, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 March 2019 Received in revised form 29 August 2019 Accepted 31 August 2019 Available online 13 September 2019

Diisononyl phthalate (DINP) is a high-molecular-weight phthalate, and has been recently introduced as di-(2-ethyl hexyl) phthalate (DEHP) substitute and commonly used in a large variety of plastic items. The fat tissue is an important target for DINP exposure, however, very little is understood about its toxicity and mechanism(s) in adipocyte cells. Therefore, the present work aimed to investigate the role of DINP in adipogenesis using 3T3-L1 preadipocytes. DINP exposure for 10 days extensively induced adipogenesis in 3T3-L1 preadipocytes to adipocytes as assessed by lipid accumulation and gene expression of adipogenic markers. The RT-qPCR results showed that DINP could upregulate the expression of peroxisome proliferator-activated receptor-gamma (PPARg), CCAAT/enhancer-binding protein alpha (C/EBPa) and C/ EBPb, while the expression of sterol regulatory element binding transcription factor 1 (SREBF1) and C/ EBPd was not affected. The DINP-induced adipogenesis could be inhibited by using the selective PPARg antagonist GW9662. The RNA-seq analysis was used to study the systemic toxicities of DINP on preadipocytes. A total of 1181 differently expressed genes (DEGs) (640 genes were up-regulated, 541 genes were down-regulated) were detected in 3T3-L1 preadipocytes under 50 mM DINP. The GO enrichment showed the GO term of “fat cell differentiation” was the most significantly affected metabolic functions, and the KEGG pathway enrichment showed the PPAR pathway was the top affected pathway. The interactive pathway (iPath) analysis showed that the changed metabolic pathways were focus on the lipid metabolism. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Diisononyl phthalate PPARg pathway Adipocyte differentiation Lipid metabolism Transcriptional profiling

1. Introduction Phthalates esters (PAEs) are a class of synthetic chemicals of multiple functions, being used to increase the flexibility, transparency, durability and longevity of industrial polymers. PAEs have been primarily applied in the manufacturing of polyvinyl chloride (PVC) and other chemical products, such as pesticides, personalcare products, printing, pharmaceuticals and lubricants (Gao and Wen, 2016). PAEs are not chemically bound to plastics and can thus leach, migrate or evaporate into the environment easily during the use. Various phthalates, such as di-(2-ethyl hexyl) phthalate (DEHP), diethyl phthalate (DEP) and dibutyl phthalate (DBP) have been detected in the environment and human body with substantial levels all over the world (Luo et al., 2018; Shi and Cao, * This paper has been recommended for acceptance by Prof. Wen Chen. * Corresponding author. E-mail address: [email protected] (H. Sun).

https://doi.org/10.1016/j.envpol.2019.113154 0269-7491/© 2019 Elsevier Ltd. All rights reserved.

2018). DEHP is historically the most commonly used phthalate in the world due to its low cost and suitable properties, being the dominant phthalates detected in environment (Zhang et al., 2018; Huo et al., 2016). However, emerging evidences have proved that DEHP exposure could increase the risk of certain adverse health outcomes, including cancer, endocrine disruption, obesity, cardiotoxicity, etc (Zhao et al., 2018a; Zhang et al., 2016; Zarean et al., 2016). Recently, DEHP has been restricted in many countries and even banned from general use under EU law in 2015, and now some substitutes are gradually replacing DEHP on the market (Larsson et al., 2017). Diisononyl phthalate (DINP) is a high molecular weight phthalate. It has been recently introduced as DEHP substitute and commonly used in a large variety of plastic items, especially as plasticizer in PVC products (Ginsberg and Belleggia, 2017). Consequently, DINP has now become one of the major phthalate compounds in the indoor (Blanchard et al., 2014; Larsson et al., 2017) and outdoor environmental media (Li et al., 2017). DINP can enter

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human bodies via dermal absorption, ingestion and inhalation (Sakhi et al., 2014), and the risks of DINP to human health are of great concern. Animal experiments have demonstrated that DINP exposure can induce reproductive toxicity (Forner-Piquer et al., 2018a,b), interfere with the immune system and aggravate some allergic diseases, including airway inflammation (Chen et al., 2015), asthma (Hwang et al., 2017), dermatitis (Kang et al., 2016), etc. Studies in zebrafish showed that DINP exposure could affect skeletal muscle composition (Carnevali et al., 2019) and the endocannabinoid system in liver and brain (Forner-piquer et al., 2018b). Toxicity mechanisms studies revealed that the DINP exposure can activate NF-kB (Duan et al., 2018; Kang et al., 2018; Kang et al., 2017), Akt/mTOR (Duan et al., 2019), PI3K/Akt pathway (Chen et al., 2015) and induce Th2 mediated cytokine (IL-4, IL-5) production (Hwang et al., 2017; Koike et al., 2010). In addition, epidemiological studies also suggested that the concentration of DINP or its primary metabolite, monoisononyl phthalate (MINP) showed a significant correlation with the serum level of thyroxine (T4) and the incidence of insulin resistance (Attina and Trasande, 2015; Huang et al., 2016). Previous studies have shown that fat tissue is a main target tissue for phthalates (Chiang et al., 2016), and their toxicity on preand mature adipocytes should be well considered. Animal studies showed that early life exposure to DEHP is potentially associated with increased adiposity in rodents (Wassenaar and Legler, 2017), and that in utero exposure to environmentally safe dose of DEHP also can lead to excessive visceral fat accumulation in C57BL/6J mice (Gu et al., 2016; Campioli et al., 2011; Qi et al., 2019). In vitro studies demonstrated that DEHP and MEHP could affect the differentiation of preadipocytes (Chiang et al., 2016), and elicit inflammatory response in adipocytes (Manteiga and Lee, 2017). Recently Pomatto et al. (2018) tested the adipogenic activity for four plasticizers, and found that DINP could increase lipid accumulation in 3T3-L1 cells. However, it is not clear yet the mechanism underlying adipogenesis and the systemic toxicities were induced by DINP. In this paper, we determined the effects of DINP on adipogenesis in 3T3-L1 preadipocytes, explored the modes of action and analyzed the systemic toxicities through global transcriptomic response to DINP. We found that DINP could induce adipogenesis by PPARg pathway. Furthermore, global gene expression analysis revealed that promoting differentiation and lipid metabolism are the primary effects for DINP exposure on preadipocytes.

isobutyl-1-methylxanthine (IBMX), dexamethason (Dex) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Rosiglitazone (Rosi) and GW9662 were purchased from APExBIO Technology (Houston, USA). Insulin (beef) was purchased from Macklin Chemical Reagent Company (Shanghai, China). Dulbecco's Modified Eagles Medium (DMEM, High glucose), Fetal Bovine Serum (FBS) were purchased from HyClone (Logan City, Utah, USA). The primary antibodies, FABP4 rabbit mAb (D25B3), b-Actin rabbit mAb (D6A8) and the HRP-linked anti-rabbit IgG (7074) were purchased from Cell Signaling Technology (Danvers, MA, USA). 2.2. Cell culture and differentiation The 3T3-L1 (ATCC CL173, lot 58432113) cells were maintained in basal medium (BM, DMEM supplemented with 10% FBS and 1% penicillinestreptomycin) in an atmosphere of 5% CO2. The 3T3-L1 preadipocytes were seeded in 6-well plates at a density of 5  105 cells/well and allowed to grow to 100% confluence with media changes every 2 days. Two days after confluence (day 0), the cells were induced to differentiate by exposing to the differentiation medium (MDI, basal medium containing 1 mg/mL insulin, 1 mM DEX, 0.5 mM IBMX). After 48 h, the MDI medium was aspirated, and the cells were fed with maintenance medium (MM, basal medium containing 1 mg/mL insulin) for 2 days. Then the cultures were maintained in basal medium and the medium was renewed every 2 days. To clarify the effects of DINP on preadipocytes, the cells were treated with series concentrations of DINP (0e100 mM) or vehicle (0.5% DMSO) during the whole period. Rosi (100 nM) was used as positive control during the whole differentiation. The timeline of the cell culture experiments was summarized in Fig. 1b. 2.3. Cell viability assay The effect of DINP on the cell viability 3T3-L1 preadipocytes was determined using cell counting kit-8 (CCK-8) (Dojindo Molecular Technologies, China). Briefly, cells were seeded in 6-well plates at density of 5  105 cells/well, 2 days after 100% confluence, the cells were treated with various concentrations of DINP for 10 days, and the medium was changed every 2 days during the exposure. At the end of exposure, approximately 100 mL of CCK-8 was added to the cells, and the OD value of the cells was measured at 450 nm using an ELISA reader (Tacan, Spark) according to the manufacturer's instructions.

2. Experimental 2.4. Oil red O staining and cellular lipid content determination 2.1. Materials Diisononyl Phthalate (DINP, mixture of C9 isomers,
99%), 3-

3T3-L1 preadipocytes were treated with DINP or Rosi for 10 days, then the supernatant was removed and cell layer was washed

Fig. 1. Schematics of (a) diisononyl phthalate and (b) exposure protocol. BM, basal medium (DMEM supplemented with 10% FBS and 1% penicillinestreptomycin); MDI, differentiation medium (basal medium containing 1 mg/mL insulin, 1 mM DEX, 0.5 mM IBMX); MM, maintenance medium (basal medium containing 1 mg/mL insulin).

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Fig. 2. The Oil red O staining results of 3T3-L1 preadipocytes in the presence of (a) control (0.5% DMSO treatment), (b) Rosi (100 nM), and DINP with concentration of (c) 1, (d) 10, (e) 50 and (f) 100 mM. DMSO, DINP or Rosi treated 3T3-L1 preadipocytes for 10 days.

twice with ice-cold PBS. Cells were fixed with 10% formalin for 1 h. Cells were washed with PBS and stained with a filtered Oil Red O solution for 1 h at room temperature. Then cells were washed and imaged under microscope. At last the Oil Red O stain per well was dissolved by adding 1.0 mL of isopropanol. Dissolved Oil Red O (0.5 mL) was transferred to a 24-well plate, and the absorbance at 520 nm was recorded using an ELISA reader (Tacan, Spark). 2.5. RT-qPCR and Western Blot analysis Total RNA was isolated using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. Two microgram of total RNA was converted to cDNA using random hexamers with FastKing RT kit (Tiangen, China). Quantitative real-time PCR was performed with the QuantStudio™ 6 and 7 Flex Real-Time PCR System (Life Technologies) using SYBR green chemistry (SuperReal PreMix Plus, Tiangen, China). The primers used are listed in Table S1. The cycle threshold (Ct) values were obtained, and the data were normalized to b-actin expression by using the DDCt method to calculate the relative mRNA level of each gene. The 3T3-L1 preadipocytes were lysed using RIPA Lysis Buffer and the protein content was measured using the BCA protein assay kit. Equal amounts of protein (15 mg per lane) were separated by SDS-

Fig. 3. Lipid accumulation in 3T3-L1 preadipocytes in the presence of Rosi (100 nM) or various concentrations of DINP (0, 1.0, 10, 50 and 100 mM). The lipid accumulation was quantified by measuring the extracted Oil Red O dye at 520 nm *P < 0.05 compared with control group (treated with DMSO).

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PAGE (12%) and then transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Germany). The membranes were blocked for 2 h with 5% non-fat milk powder in TBST (TBS containing 0.05% Tween 20) and then incubated over night with the primary antibody at a dilution of 1:1000 at 4  C. After washed with TBST, the membranes were incubated with HRP-linked secondary antibody at a dilution of 1:1000 for 1 h. Then the membranes were washed and the bound antibody signal was developed using an Immun-Star HRP chemiluminescent kit. The Immunoblot images were captured on the Chemiluminescence Imaging System (Tanon 5200, Shanghai, China) and quantified using Image J program (NIH). 2.6. Reporter gene assay HEK-293T cells were maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics in an atmosphere of 5% CO2. HEK-293T cells were seeded at a density of 1  105 cells/well in a 24-well plate and were cotransfected with pBIND-hPPARg-LBD expression plasmid and pGL4.35 [luc2P/9XGAL4UAS/Hygro] plasmid using Lipofectamine 2000 (Invitrogen) by following the manufacturer's protocol. After exposure to the transfection reagents and plasmids for 6 h, the cells were washed with PBS and then incubated with the medium containing different

concentrations of DINP or Rosi (100 nM) for 24 or 48 h. Then the cells were washed with PBS and harvested with Passive Lysis Buffer. Firefly luciferase (reporter pGL4.35) and Renilla luciferase (pBIND) activities were determined using the Dual luciferase assay system kit (Promega) according to manufacturer's instructions. 2.7. cDNA library construction and sequencing High-quality RNA obtained using TRIzol reagent was used to enrich poly(A) mRNA using oligo (dT) magnetic beads. The obtained mRNA was fragmented into small pieces by fragmentation buffer. The first strand cDNA was synthesized using random Hexamer Primer and reverse transcriptase (Invitrogen) and the secondstrand cDNA was synthesized using RNaseH and DNA polymerase. The synthesized dscDNA was subjected to end-repair and 30 adenylation. The adaptors with “T” at 3’ end were ligated to the dscDNA, and these ligation products were used for PCR amplification. The PCR products were heat denatured, and the singlestranded DNA was cyclized by splint oligo and DNA ligase to format as the cDNA library. Then, the library was used for RNA sequencing (RNA-seq) on the sequencing platform, BDISEQ-500 (BGI, Shenzhen, China) with a read length of 50 bp. All the generated raw sequencing reads were filtered to remove reads with

Fig. 4. Effects of DINP on FABP4 (a) mRNA and (b) protein expression. 3T3-L1 preadipocytes were exposed under DINP or Rosi (100 nM) for 10 days. The mRNA levels of were quantified by real-time qPCR, and the protein levels were determined by western-blot, normalized to endogenous b-actin, and expressed as a fold over the control. *P < 0.05 compared with the control (treated with DMSO).

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adaptors, reads in which unknown bases were more than 10%, and low-quality reads. Clean reads were then obtained and stored in FASTQ format. Bowtie 2 was used to map clean reads to reference genes, and HISAT was used to reference the genome. The sequence reads have been submitted to NCBI-SRA database (Temporary Submission ID: SUB5257630). 2.8. Gene expression analysis and enrichment analysis Expression levels for each of the genes were normalized to fragments per kilobase of exon model per million mapped reads (FPKM) using RNA-seq by Expectation Maximization (RSEM). The DEGs (differential expressed genes) were identified between samples and performed clustering analysis and functional annotation. Genes with
2-fold change and false discovery rate (FDR) of 0.05 were considered to be statistically significant. Blast2GO was used for functional categorization of the DEGs. The GO enrichment analysis of DEGs was performed by GOAtools, and the KEGG pathway analysis was performed using the phyper function within the R platform based on KEGG pathway annotation. The GO terms and KEGG pathways with FDR 0.05 were considered significantly enriched in DEGs. The enrichment ratio was calculated as the number of DEGs enriched in this term (pathway), to the number of

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all genes annotated in the same pathway. 2.9. Statistical analyses All experiments were independently performed three times in triplicate and the results were expressed as the mean ± SD. Multiple comparisons were performed by one-way analysis of variance (ANOVA), and statistical significance for pairwise comparison was determined by Dunnett's post test. A p-value <0.05 was considered statistically significant. 3. Results 3.1. DINP induce 3T3-L1 preadipocytes differentiation To determine whether DINP has growth-inhibitory effects in 3T3-L1 preadipocytes, the cells were exposed to different concentrations of DINP for 10 days. As shown in Fig. S1, the DINP has no obvious cytotoxicity on the 3T3-L1 preadipocytes in the tested concentration of 0e200 mM. The DINP exposure induced the morphological change and formed lipid droplet in the cultured cells (Fig. S2). After staining with Oil Red O, the DINP exposed cells, as well as the positive

Fig. 5. PPARg mediated the DINP induced adipogenesis. (a) Effects of DINP on PPARg mediated luciferase reporter gene transcription activity. The luciferase reading was normalized to the control (DMSO treated) at the corresponding time point. *P < 0.05 compared with the control at the corresponding time point. Effect of GW9662 on DINP- or Rosi-induced (b) FABP4 mRNA upregulation and (c) lipid formation. 3T3-L1 preadipocytes treated with either DMSO, DINP (50 mM) or Rosi (100 nM) alone or co-treated with GW9662 (100 mM) for 10 days. The mRNA levels were expressed as a fold over the control (DMSO treatment). *P < 0.05.

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control (treated with Rosi), accumulate lipid in spherical cellular inclusions, indicating that DINP enhanced chemically induced adipogenesis (Fig. 2). To quantify the lipid accumulation, Oil Red O was extracted by adding isopropanol, and the results showed that the 3T3-L1 preadipocytes treated with DINP exhibited a dosedependent increase in lipid accumulation, with statistical significance achieved at 10 mM (Fig. 3).

3.2. DINP exposure upregulates FABP4 mRNA and protein expression To further assess the extent of adipocyte differentiation and to delineate whether in 3T3-L1 preadipocytes DINP upregulate the expression in mRNA and protein levels of the mature adipocyte marker, fatty acid binding protein 4 (FABP4) were measured by RTqPCR and Western Blot analysis, respectively. As shown in Fig. 4,

Fig. 6. Gene expression analysis of transcriptional regulators involved in 3T3-L1 differentiation in undifferentiated (Undiff control) or differentiated cells following exposure to DMSO (Diff control), DINP (50 mM) and Rosi (100 nM) at various time points during differentiation. RNA was isolated and the mRNA levels were quantified by real-time qPCR. Levels were normalized to the differentiated control of day 0. # Rosi significantly different compared to differentiated control at the same exposure time, * DINP significantly different compared to differentiated control at the same exposure time, P < 0.05.

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DINP significantly increased FABP4 expression, and an increasing trend was observed with DINP concentration in both mRNA and protein levels. The highest expression was observed at 50 mM of DINP exposure, which induced the FABP4 mRNA increasing by ~47fold compared with the control, and importantly, the FABP4 upregulation was observed even at low DINP concentration of 1.0 preadipocytes (p < 0.05). Importantly, as shown in Fig. S3, the FABP4 mRNA was significantly upregulated at days 2 when exposed to10 and 50 mM DINP. We also measured the expression of adiponectin (Adipoq), lipoprotein lipase (Lpl), and fatty acid synthase (Fasn), which were reported to be used as adipogenesis markers, and the results showed that the DINP exposure induced the upregulation of Adipoq and Lpl, however, unlike Rosi, the Fasn was not significantly affected with DINP exposure (Fig. S4). We also investigated whether DINP is sufficient to induce differentiation by itself. The 3T3-L1 preadipocytes were exposed to DINP (100 mM) or Rosi (100 nM) for 10 days in the absence of IBMX, DEX, and insulin. The results in Fig. S5 showed that DINP, as well as Rosi exposure slightly increased the FABP4 expression, but neither of them significantly induced lipid accumulation. 3.3. PPARg mediated the DINP induced adipogenesis The observed increase in adipocyte differentiation by DINP in 3T3-L1 cells led us to verify the possibility that DINP may be a ligand for PPARg. As shown in Fig. 5a, DINP showed no obvious PPARg activating effect at the concentration of 0e150 mM and the PPARg was activated about 1.5 folds at the DINP concentration of 200 mM under 24 h exposure. For 48 h exposure, DINP exhibited PPARg activation at the concentration of 10e200 mM and the luciferase transcriptional activity was enhanced about 1.28, 1.40 and 1.72 folds at the DINP concentrations of 10, 100 and 200 mM, respectively. Overall, DINP manifested a concentration- and timedependent PPARg activation. However, the activation of PPARg induced by DINP was much lower compared to those induced by Rosi (100 nM) which activated PPARg by 10.3 and 27.4 folds under 24 and 48 h treatment, respectively (Fig. S6). To investigate the involvement of PPARg in the adipogenesis elicited by DINP, we repeated the exposure experiments in the presence of the selective PPARg antagonist GW9662. As shown in Fig. 5b and c, the FABP4 upregulation and lipid formation induced by DINP were abolished with co-treatment of GW9662. As expected, the GW9662 also suppressed the differentiation induced by the specific PPARg agonist Rosi in 3T3-L1 preadipocytes. This indicates that PPARg mediates DINP induced adipogenesis in 3T3-L1 preadipocytes.

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went down to the same level of the differentiated control. 3.5. Transcriptional profiling of 3T3-L1 preadipocytes exposure to DINP Under our experimental conditions, a total of 1181 DEGs (640 genes were up-regulated, 541 genes were down-regulated) were detected in 3T3-L1 preadipocytes exposed to 50 mM DINP compared with the vehicle control, using two fold change (pvalue < 0.05) as the threshold (Fig. 7). The gene expression of Lpl, Adipoq, Fasn, Adipor1 and Tnc (tenascin C) were validated using RTqPCR, and showed that the results of RNA-seq and RT-qPCR were well-matched (Table S2). The DEGs were allocated to three primary GO categories known as biological process, cellular component and molecular functions, which were subsequent assigned to 54 functional terms (Table S3, with the top 20 GO categories listed in Fig. S7). Within the “biological process” category, most DEGs were assigned to “single-organism process (GO: 0044699)”, “cellular process (GO: 0009987)”, “metabolic process (GO: 0008152)”, “biological regulation (GO:0051704)” and “regulation of biological process (GO: 0050789)”. Within the “cellular component” category, “cell (GO: 0005623)”, “cell part (GO: 0044464)” and “organelle (GO: 0043226)” were the most primary subcategories. As for the “molecular function”category, the mainly enriched subcategories were “binding (GO: 0005488)” and “catalytic activity (GO: 0003824)”. 3.6. GO and KEGG enrichment analysis To better understand the biological functions affected by DINP exposure, we further performed enrichment analysis based on GO and KEGG annotation of the DEGs. Results of GO enrichment analysis were summarized in Fig. 8, the GO terms of “fat cell differentiation (GO: 0045444) and “brown fat cell differentiation (GO: 0050873)” were the most significantly affected metabolic functions. Along with the adipocyte differentiation and lipid accumulation, the GO terms of “cellular lipid metabolic process” and “lipid metabolic process” were also enriched significantly. Four pathways were significantly enriched using KEGG pathway enrichment analysis, and the PPAR signaling pathway (Pathway id: map03320)

3.4. Effect of DINP on the transcriptional regulators of adipogenesis Because the DINP showed only weak activation of PPARg, we assessed whether DINP promoted adipogenesis through the upregulation of genes known to be involved in adipogenic differentiation and/or lipid metabolism. In this study, the key regulator genes involved in 3T3-L1 adipogenesis, PPARg, C/EBPa, C/EBPb, C/EBPd and SREBF1 were measured at various time points during induced differentiation by DINP or Rosi exposures (Fig. 6). When compared with the differentiated control, the mRNA levels of these five regulators were all upregulated by Rosi during the whole or partial differentiation period. After exposure to DINP (50 mM), the mRNA level of PPARg was upregulated during the whole differentiation period, and C/EBPa was upregulated at days 6e10. However, the mRNA levels of C/EBPd and SREBF1 were not affected during the whole differentiation process. Interestingly, the expression of C/ EBPb was increased in response to DINP at the first 4 days and then

Fig. 7. Scatter plots of all expressed genes in the control and DINP exposure groups. Cells were treated with DINP (50 mM) and control (DMSO) during the whole differentiation period. Genes with
2-fold change and false discovery rate (FDR) of 0.05 were considered to be statistically significant.

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Fig. 8. The (a) GO and (b) KEGG pathway enrichments of the DEGs. The GO terms and pathways were arranged left-to-right according to FDR values. *** FDR<0.001, ** FDR<0.01 and * FDR<0.05.

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Fig. 9. iPath analysis of the DEGs under DINP exposure. DEGs were mapped onto the lipid metabolism map. The red lines indicate upregulated metabolic pathways.

was the most significant enriched pathway (FDR<0.001), and the regulation of lipolysis in adipocytes (Pathway ID: map00561) was also enriched significantly. 3.7. iPath metabolic pathway analysis The DEGs were used to map to iPath to investigate their metabolism relationships and to further understand the effects of DINP on the metabolism of 3T3-L1 preadipocytes. The results indicated that DINP-induced DEGs were non-randomly clustered among nucleotide metabolism, lipid metabolism, pyruvate metabolism, carbohydrate metabolism, and oxidative phosphorylation, etc. As can be seen from Fig. S8, the lipid metabolism and nucleotide metabolism were the two most significantly affected metabolic categories. Further analysis showed that the lipid metabolism and nucleotide metabolism were up- and down-regulated under DINP exposure, respectively. Analysis of the map in lipid metabolism

category indicated that several lipid metabolic pathways were enriched, which included “fatty acid biosynthesis”, “fatty acid elongation in mitochondria”, “fatty acid metabolism” and “butanoate metabolism” (Fig. 9). This indicated that the lipid metabolism was the most changed metabolism pathway under DINP exposure. 4. Discussion This study demonstrates for the first time that promoting cell differentiation and lipid metabolism are the primary effects for DINP exposure on 3T3-L1 preadipocytes. Obesity is a serious medical condition that can cause many health problems such as type 2 diabetes, insulin resistance, atherosclerosis and stroke (Ou et al., 2019; Liu and Ding, 2017). Over the past two decades, the prevalence of obesity and obesity-associated metabolic diseases had been dramatically increased globally (Zhao et al., 2018b). Adipocyte hyperplasia is a critical event for the development of

Table 1 Fatty cell differentiation-related DEGs identified by GO enrichment. Gene ID

Gene Name

Gene Description

FC (log2)

FDR

ENSMUSG00000034957 ENSMUSG00000039886 ENSMUSG00000030545 ENSMUSG00000051314 ENSMUSG00000037071 ENSMUSG00000012428 ENSMUSG00000023034 ENSMUSG00000029322 ENSMUSG00000039956 ENSMUSG00000018566 ENSMUSG00000037035 ENSMUSG00000070348 ENSMUSG00000062515 ENSMUSG00000071657 ENSMUSG00000009281 ENSMUSG00000045658 ENSMUSG00000044405 ENSMUSG00000026360 ENSMUSG00000054434 ENSMUSG00000022878

Cebpa Tmem120a Pex11a Ffar2 Scd1 Steap4 Nr4a1 Plac8 Mrap Slc2a4 Inhbb Ccnd1 Fabp4 Bscl2 Rarres2 Pid1 Adig Rgs2 Tmem120b Adipoq

CCAAT/enhancer binding protein (C/EBP), alpha transmembrane protein 120A peroxisomal biogenesis factor 11 alpha free fatty acid receptor 2 stearoyl-Coenzyme A desaturase 1 STEAP family member 4 nuclear receptor subfamily 4, group A, member 1 placenta-specific 8 melanocortin 2 receptor accessory protein solute carrier family 2, member 4 inhibin beta-B cyclin D1 fatty acid binding protein 4, adipocyte Berardinelli-Seip congenital lipodystrophy 2 (seipin) retinoic acid receptor responder (tazarotene induced) 2 phosphotyrosine interaction domain containing 1 adipogenin regulator of G-protein signaling 2 transmembrane protein 120B adiponectin, C1Q and collagen domain containing

3.55144041 1.7291489 2.002099631 3.20534805 1.682710111 1.328305349 1.108313984 1.064338315 4.245021366 3.957677394 1.263376111 1.068099841 4.679931647 2.514319277 3.338618125 1.012702293 4.461662882 1.228827551 2.657903767 4.570573886

3.01E-43 3.97E-08 4.01E-09 4.85E-11 7.56E-14 0.028864799 0.000941185 0.000950507 4.38E-40 2.08E-37 2.03E-05 0.000239084 6.88E-96 2.32E-13 1.59E-31 0.016345752 3.93E-47 5.97E-05 1.15E-19 3.89E-91

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obesity and arises due to adipogenic differentiation of preadipocytes (Bost et al., 2005). The 3T3-L1 cell line is a widely used model system for the study of preadipocytes differentiation (Cave and Crowther, 2019). Promoting adipocyte differentiation is one of the toxicities for some environmental pollutants, such as MEHP (Chiang et al., 2016), perfluorooctane sulfonate (PFOS) (Xu et al., 2016), Bisphenol A (Boucher et al., 2015), p,p'-dichlorodiphenyldichloroethylene (DDE) (Mangum et al., 2015), chlorinated polyfluorinated ether sulfonates (Li et al., 2018), etc (Thayer et al., 2012). In this study, we found that the DINP exposure could change the cells morphology and form lipid drops. The adipogenesis was further confirmed through the upregulation of terminal differentiation makers of FABP4, Lpl and Adipoq. FABP4, also known as adipocyte P2 (aP2), is expressed primarily in adipocytes, and regarded as an adipogenic marker. The function of FABP4 includes fatty acid uptake, transport and metabolism, and is essential for fatty acid-induced lipid droplet accumulation (Scifres et al., 2011). Adipoq is a protein hormone and regulates lipid catabolism and insulin sensitivity, and Adipoq can upregulate uncoupling protein (UCP) to disturb energy metabolism (Bauche et al., 2007). Interestingly, we observed the upregulation of UCP-2 (gene id: 22228) and energy disrupting in RNA-seq analysis, indicating the Adipoq may mediate many other toxicities induced by DINP. Adipogenesis is a process that tightly controlled by an intricate network of transcription factors (Farmer, 2006). Several studies have clearly established that PPARg and C/EBPa are master regulators in adipogenesis, and the upstream regulators of C/EBPb, C/ EBPd and SREBP-1 also play an important role in this process (Sarjeant and Stephens, 2012). The gene-knockout study showed that PPARg could induce adipogenesis in C/EBPa/ mouse embryonic fibroblasts, whereas the C/EBPa is unable to do the same in PPARg/ models (Rosen et al., 2002). This study showed that DINP could weakly activate PPARg in transient transfection assays. The activation of PPARg is likely to be causally related to the adipogenesis and many PPARg target genes after exposure to DINP. We also investigated other key regulators in 3T3-L1 adipogenesis, and found that PPARg, C/EBPa and C/EBPb were upregulated by DINP exposure, while neither C/EBPd nor SREBP-1 was affected. As expected, in the positive control, all of the five transfactors were upregulated. This further indicates that DINP and Rosi, although both may promote adipogenesis, their modes of action may be different. Further, the lipogenesis could be inhibited in the presence

of the selective PPARg antagonist GW9662. Taken together, the PPARg pathway mediated DINP to induce adipogenesis. To further investigate the systemic toxicities of DINP on 3T3-L1 preadipocytes, we used RNA-seq to analyze the transcriptome changing profiles. To our knowledge, this is the first study to compare global transcriptomic changes in response to DINP in preadipocytes. GO enrichment analysis revealed that DINP mainly influenced the fat cell differentiation, in which a total of 20 DEGs were annotated, including 17 genes upregulated and 3 genes downregulated (Table 1). In the KEGG pathway analysis, the PPAR pathway (map03320), valine, leucine and isoleucine degradation (map00280), retrograde endocanabinoid signaling (map04723) and glycerophospholipid metabolism (map00564) were listed in the significantly affected pathways under DINP treatment. Considering the enrichment ratio and FDR values, the PPAR signal pathway was the most significantly affected pathway. Further analysis showed that there are 21 DEGs related with PPAR pathway (Table 2). The 20 up-regulated DEGs are involved in lipid uptake (cd36), lipid binding and transport (fabp4, fabp5, acbp, cd36, scp2), lipid synthesis (acaa1a, scd1, acsl1), and lipid storage (plin1). We found that, along with the lipid accumulation, the lipolysis (Lpl) was also promoted under DINP exposure. In addition to adipocyte differentiation and lipid metabolism, the PPARg also plays an import role in inflammation, we found that the chemokine (C-C motif) ligand 2 (ccl2, Gene ID: 20296), a major chemotactic factor that signals for recruitment and M1 polarization of macrophages, was down-regulated, indicating that the DINP exposure may disrupt the inflammation in preadipocytes though PPARg. The lipid metabolism is a complex process and participates in the regulation of many cellular processes such as cell growth, apoptosis, inflammation and membrane homeostasis (Huang and Freter, 2015). In the iPath metabolic map, the changed metabolic pathways were focus on the lipid metabolism, including fatty acid biosynthesis, metabolism, and fatty acid elongation in mitochondria and butanoate metabolism. In the present study, the effect of DINP on preadipocytes was evaluated by analyzing the DEGs between DINP exposure and the control. Compared with rosiglitazone, DINP is a weaker PPARg agonist, so its systemic effect on preadipocytes is speculatively different to that induced by Rosi. It would have been interesting to learn which of the DEGs are also up- or down-regulated by Rosi and are also known targets of PPARg-mediated regulation.

Table 2 PPAR signaling-related DEGs identified by KEGG pathway analysis. Gene ID

Gene Name

Gene Description

FC (log2)

FDR

ENSMUSG00000002831 ENSMUSG00000032081 ENSMUSG00000030546 ENSMUSG00000036138 ENSMUSG00000037071 ENSMUSG00000031278 ENSMUSG00000018796 ENSMUSG00000074280 ENSMUSG00000028603 ENSMUSG00000025006 ENSMUSG00000027533 ENSMUSG00000062515 ENSMUSG00000028427 ENSMUSG00000002944 ENSMUSG00000028607 ENSMUSG00000015568 ENSMUSG00000015843 ENSMUSG00000026385 ENSMUSG00000022878 ENSMUSG00000002108 ENSMUSG00000027513

Plin4 Apoc3 Plin1 Acaa1a Scd1 Acsl4 Acsl1 Gm6166 Scp2 Sorbs1 Fabp5 Fabp4 Aqp7 Cd36 Cpt2 Lpl Rxrg Acbp Adipoq Nr1h3 Pck1

perilipin 4 apolipoprotein C-III perilipin 1 acetyl-Coenzyme A acyltransferase 1A stearoyl-Coenzyme A desaturase 1 acyl-CoA synthetase long-chain family member 4 acyl-CoA synthetase long-chain family member 1 predicted gene 6166 sterol carrier protein 2, liver sorbin and SH3 domain containing 1 fatty acid binding protein 5, epidermal fatty acid binding protein 4, adipocyte aquaporin 7 CD36 antigen carnitinepalmitoyltransferase 2 lipoprotein lipase retinoid X receptor gamma Acyl-CoA-binding protein adiponectin, C1Q and collagen domain containing nuclear receptor subfamily 1, group H, member 3 phosphoenol pyruvatecarboxykinase 1, cytosolic

2.515810569 2.312848982 4.574670894 1.005646758 1.682710111 1.080822806 3.597542255 1.034730048 1.435750164 1.929830948 1.061205904 4.679931647 4.049845004 2.903058048 1.253414428 1.473235939 2.768752656 1.76268395 4.570573886 2.376763287 2.880460322

8.17177E-30 4.19482E-05 8.70403E-83 0.001004457 7.55789E-14 0.003431185 3.1884E-63 0.005580331 1.07364E-08 4.13451E-08 0.001006157 6.88338E-96 4.60686E-25 5.38139E-23 3.56714E-06 1.7124E-09 7.18687E-08 2.56327E-13 3.88593E-91 2.39858E-14 1.96019E-10

L. Zhang et al. / Environmental Pollution 255 (2019) 113154

In conclusion, we demonstrated that the DINP promotes adipogenesis in 3T3-L1 preadipocytes in vitro. DINP can induce adipogenesis via the PPARg pathway, and the global gene expression profiles revealed that the PPARg pathway and lipid metabolism are the most significantly affected function under DINP exposure. Overall, the results of this study suggest that exposure to DINP, which is considered to be less toxic and used as a substitute for DEHP, may affect normal physiological functions and therefore, further investigation into DINP-induced other toxicity, e.g. mitotic cell cycle process (GO:1903047), which is the main changes for the down-regulated DEGs, is needed. Conflicts of interest There are no conflicts of interest. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 21607021), China Postdoctoral Science Foundation (No. 2017M621058) and National Natural Science Foundation of China (No. 21707099). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.113154. References Attina, T.M., Trasande, L., 2015. Association of exposure to di-2-ethylhexylphthalate replacements with increased insulin resistance in adolescents from NHANES 2009-2012. J. Clin. Endocrinol. Metab. 100, 2640e2650. Bauche, I.B., El Mkadem, S.A., Pottier, A.M., Senou, M., Many, M.C., Rezsohazy, R., Penicaud, L., Maeda, N., Funahashi, T., Brichard, S.M., 2007. Overexpression of adiponectin targeted to adipose tissue in transgenic mice: impaired adipocyte differentiation. Endocrinology 148, 1539e1549. Blanchard, O., Glorennec, P., Mercier, F., Bonvallot, N., Chevrier, C., Ramalho, O., Mandin, C., Bot, B.L., 2014. Semivolatile organic compounds in indoor air and settled dust in 30 French dwellings. Environ. Sci. Technol. 48, 3959e3969.
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