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Sub-chronically exposing mice to a polycyclic aromatic hydrocarbon increases lipid accumulation in their livers Yuanxiang Jin 1 , Wenyu Miao 1 , Xiaojian Lin, Tao Wu, Hangjie Shen, Shan Chen, Yanhong Li, Qiaoqiao Pan, Zhengwei Fu ∗ College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China

a r t i c l e

i n f o

a b s t r a c t

Article history:

The potential for exposing humans and wildlife to environmental polycyclic aromatic

Received 7 May 2014

hydrocarbons (PAHs) has increased. Risk assessments describing how PAHs disturb lipid

Received in revised form

metabolism and induce hepatotoxicity have only received limited attention. In the present

11 July 2014

study, seven-week-old male ICR mice received intraperitoneal injections of 0, 0.01, 0.1 or

Accepted 19 July 2014

1 mg/kg body weight 3-methylcholanthrene (3MC) per week for 10 weeks. A high-fat diet was

Available online 28 July 2014

provided during the exposure. Histopathological lipid accumulation and lipid metabolism-

Keywords:

increased lipid droplet and triacylglycerol (TG) levels in the livers. A low dose of 3MC acti-

Polycyclic aromatic hydrocarbon

vated the aryl hydrocarbon receptor, which negatively regulated lipid synthesis in the livers.

Sub-chronic exposure

The primary genes including acetyl-CoA carboxylase (Acc), fatty acid synthase (Fas) and stearoyl-

related genes were measured. We observed that sub-chronic 3MC exposure significantly

Lipid metabolism

CoA desaturase 1 (Scd1) decreased significantly when compared with those in the control

Gene transcription

group, indicating that de novo fatty acid synthesis in the hepatocytes was significantly inhibited by the sub-chronic 3MC exposure. However, the free fatty acid (FFA) synthesis

Mice

in the adipose tissue was greatly enhanced by up-regulating the expression of peroxisome proliferator-activated receptor ␥ (PPAR␥) and sterol regulatory element binding protein-1c (SREBP1C) and target genes including Acc, Fas and Scd1. The synthesized FFA was released into the blood and then transported into the liver by the up-regulation of Fat and Fatp2, which resulted in the gradual accumulation of lipids in the liver. In conclusion, histological examinations and molecular level analyses highlighted the development of lipid accumulation and confirmed that 3MC significantly impaired lipid metabolism in mice. © 2014 Elsevier B.V. All rights reserved.

1.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a family of persistent and hydrophobic environmental toxins that originate

from the incomplete combustion of carbon-based fuels and some carbon-containing fuels, such as wood, coal, diesel, fat and tobacco, among others (Van-Metre and Mahler, 2005; Weisman et al., 2010). Measurable and relatively high levels of different PAHs have been observed around the world

∗ Corresponding author at: College of Biological and Environmental Engineering, Zhejiang University of Technology, 18, Chaowang Road, Hangzhou, China. Tel.: +86 571 8832 0599; fax: +86 571 8832 0599. E-mail address: [email protected] (Z. Fu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.etap.2014.07.014 1382-6689/© 2014 Elsevier B.V. All rights reserved.

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in urban atmospheres, surface water, sediment, soil, plants and even in different organisms, such as fish, in recent years (Klumpp et al., 2002; Wang et al., 2006; Li et al., 2009, 2012; Korosi et al., 2013). More importantly, PAHs can enter the body from various sources including coal, coke and diesel fuel burning, cooking activities, grilled meats, cigarettes and also from the suspended particulate matter in the air (Bonner et al., 2005; Wang et al., 2013). As a result, wildlife and humans will suffer from either observable or imperceptible consequences by PAHs exposure. A number of previous studies have indicated that PAHs could be activated as ligands to bind with aryl hydrocarbon receptors (AHR) (Mimura and Fujii-Kuriyama, 2003; Ovesen et al., 2011). AHRs belong to the basic helixloop-helix/Per-Arnt-Sim (bHLH/PAS) family. In the absence of a ligand, the AHR is sequestered in the cytosol by two heat-shock protein 90 molecules. As ligands, PAHs can competitively bind to AHR and are then translocated into the nucleus. In the nucleus, they heterodimerize with the AHR nuclear translocator (ARNT) and recognize their cognate DNAbinding site, the xenobiotic response element (XRE), which is located in the regulatory regions of AHR-responsive genes, leading to the activation of target gene transcription (the most well-characterized genes are cyp1a1 and cyp1b1) (Abel and Haarmann-Stemmann, 2010; Wang et al., 2004; Beedanagari et al., 2010). PAHs have been of great concern in recent years because of their carcinogenic properties, which are very harmful to humans and wildlife. In addition to their carcinogenic properties, the roles of PAHs in hepatotoxicity and hepatic steatosis have also caused great concern in recent years (Kawano et al., 2010; Angrish et al., 2011). Hepatic lipid metabolism is known to be regulated by a variety of pathways. Previous studies have indicated that the AHR-mediated pathway was also involved in the hepatic lipid metabolism process. Sato et al. (2008) used a microarray assay to reveal that low-dose 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) treatment altered the transcription of genes related to cholesterol biosynthesis, lipogenesis and glucose metabolism in the mouse liver, suggesting that the AHR is involved in regulating cholesterol and lipid metabolism. Tanos et al. (2012) reported that AHR activation repressed the expression of hepatic fatty acid synthesis genes including acetyl-CoA carboxylase (Acc), fatty acid synthase (Fas) and stearoyl-CoA desaturase 1 (Scd1) in the livers of C57BL/6J mice. As agonists of AHRs, PAHs and dioxins may therefore play very important roles in hepatic lipid metabolism. The liver is the major lipogenesis tissue. The triacylglycerol (TG) content of hepatocytes is generally regulated by the activity of cellular molecules that facilitate hepatic free fatty acid (FFA) uptake, fatty acid synthesis, esterification and hepatic fatty acid oxidation and TG export (Nguyen et al., 2008). Thus, both the enhanced synthesis and uptake of fatty acids would lead to the accumulation of lipids in the liver. At the molecular level, hepatic fat metabolism is regulated by an abundance of key transcription factors such as peroxisome proliferatoractivated receptors ␣ and ␥ (PPAR␣ and ␥) and sterol regulatory element binding protein-1c (SREBP1C), which mediate the expression of target genes related to fatty acid synthesis including Acc, Fas, Scd1, glyceraldehyde 3-phosphate acyltransferase (Gpat) and the expression of genes related to fatty acid oxidation including carnitine palmitoyltransferase-1␣ (Cpt1␣) and

hormone-sensitive lipase (Hsl) (Jump et al., 2005; Nguyen et al., 2008; Hagiwara et al., 2012). Previous studies indicated that PAH exposure disturbed lipid metabolism by influencing different endpoints. For example, Kawano et al. (2010) reported that a single intraperitoneal injection of 100 mg/kg BW 3methylcholanthrene (3MC) enhanced the expression level of PPAR␣ and fatty acid translocase (FAT). The authors indicated that 3MC induced hepatic microvesicular steatosis by increasing the expression level of FAT. However, the mechanism through which PAH exposure disturbs lipid metabolism has not been fully elucidated in a mammalian system. In the present study, we focused on lipid metabolism in mouse livers followed by exposure to low-dose 3MC for a long period of time. Adult male ICR mice were injected with 0.01, 0.1 and 1 mg/kg BW/week 3MC, which is a common isomer of methylcholanthrene, for 10 weeks. We then analyzed the hepatic and serum parameters related to fat and cholesterol metabolism including the TG, TC, FFA and VLDL levels. Additionally, the quantity of lipid droplets in the hepatocytes was determined by cryosection to illustrate the lipid accumulation induced by sub-chronic 3MC exposure. The mRNA levels of genes related to fatty acid synthesis, oxidation and transport were then further determined to elucidate the potential mechanism underlying the lipid metabolism disruption induced by this PAH at a low dose over a long period of exposure. All the information acquired in this study is intended to provide new insights into how PAHs induce the sub-chronic mammalian toxicity.

2.

Materials and methods

2.1.

Chemicals

The original 3MC (CAS No.: 56-49-5, purity: 99.9%) was purchased from Supelco (Bellefonte, USA) and dissolved in corn oil (Wako, Japan) before injection.

2.2.

Animals and experimental design

A total of 30 6-week-old male ICR mice were purchased from the China National Laboratory Animal Resource Center (Shanghai, China). The mice were kept in our animal facilities (illuminated with strip lights to shine 200 lx at cage level with a photoperiod of 12 h light and 12 h dark; 22 ± 1 ◦ C) for 1 week prior to the experiments. Water and food were available ad libitum. The mice were then randomly divided into 5 groups. One group was given a basal diet (BD) and defined as the BD group. The remaining 4 groups were fed a high-energy diet (HD). For standardizing the intake volume according to their bodyweights, intraperitoneal injection was adopted in the experiment. The 4 groups of mice received intraperitoneal injections of 0, 0.01, 0.1 or 1 mg/kg body weight (BW) 3MC per week for 10 weeks, and the groups were named HD, HD-0.01, HD-0.1 and HD-1, respectively. During the exposure, the BD and HD groups were injected with the same volume of corn oil without 3MC. The composition of the BD was previously described, and the mineral and vitamin mix was prepared according to AIN-76 (Bieri, 1979). The HD treatment was prepared by adding 10% sucrose (wt/wt), 25% lard (wt/wt) and 1%

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 353–363

cholesterol (wt/wt) to the BD. The body weight of each mouse was measured every week during 3MC administration. After 10 weeks exposure, all the mice were sacrificed at the 7th day after the last time of intraperitoneal injection of 3MC. The liver and epididymal fat tissues were quickly removed and weighed. Partial samples from the livers were collected from 2 randomly selected mice in each group, and the samples were embedded in O.C.T. compound (Sakura Finetek, USA) and frozen in dry ice. Partial samples of the liver and epididymal fat were directly fixed in a 4% paraformaldehyde solution for histological analysis. The remaining liver and epididymal fat tissues were stored at −80 ◦ C for further use. Blood was collected, and the plasma was separated via centrifugation at 4 ◦ C and stored at −20 ◦ C until use. Every effort was made to minimize animal suffering during each experiment. All experiments were performed in accordance with the Guiding Principles in the Use of Animals in Toxicology from Zhejiang University of Technology.

2.3.

Hepatic lipids and histopathological analysis

Cryosectioned liver samples were cut into 10 ␮m slices on a cryostat (Microtome Cryostat HM 550, Walldorf, Germany) and stained with Oil red O to determine the hepatic lipid contents. Livers and epididymal fats fixed in 4% paraformaldehyde solution were sequentially processed in ethanol, xylene and paraffin. Tissues were then embedded in paraffin wax, sectioned (4 ␮m) with a Leica RM2235 (Germany) and mounted on slides. The sections were then stained with hematoxylin and eosin (H&E) prior to microscopic examination (Olympus, Japan).

2.4. Determinations of serum TG, TC, FFA and VLDL and hepatic TG and TC levels The serum TG, TC, FFA and VLDL contents were determined by using kits from the Nanjing Jianchen Institute of Biotechnology (Nanjing, China) according to the manufacturer’s instructions. For the hepatic TG and TC levels, the liver tissue was homogenized with 3 volumes of methanol and 6 volumes of chloroform were then added; the tissue was extracted for 16 h at room temperature. The chloroform layer was then collected by centrifugation at 3000 × g for 10 min. A 10 ␮l portion of each sample was used to determine the TG and TC levels with commercial kits.

2.5.

mRNA quantification

Total RNA was isolated from the liver and epididymal fat with TRIzol reagent (Takara Biochemicals, Dalian, China), and cDNA was synthesized using a reverse transcriptase kit (Toyobo, Japan). A real-time quantitative polymerase chain reaction (RT-qPCR) was performed in an Eppendorf MasterCycler ep RealPlex2 (Wesseling-Berzdorf, Germany). All the oligonucleotide primers are listed in Table S1. The 18S RNA transcript level was determined for use as a housekeeping gene. The following PCR protocol was adopted: denaturation for 1 min at 95 ◦ C, followed by 40 cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C. The PCR protocol and relative gene expression quantification were performed among the treatment groups according to our previous description (Jin et al., 2012).

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Supplementary Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.etap. 2014.07.014.

2.6.

Western blot analysis

Total liver and epididymal fat proteins were isolated with a commercial RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and protease inhibitors) (Beyotime Institute of Biotechnology, China) according to the manufacturer’s protocol. SDS-polyacrylamide gel electrophoresis was conducted as previously described (Laemmli, 1970). Proteins in the gels were electrophoretically separated by 30 mA for the first 30 min and then by 40 mA until the end of the electrophoresis. Proteins were separated via 12% SDS-PAGE, and the proteins of interest were then detected using PPAR˛ Santa Cruz Biotechnology, sc-9000, PPAR (Santa Cruz Biotechnology, sc-7273) and SREBP1 (Santa Cruz Biotechnology, sc-366) antibodies according to the manufacturer’s protocol. The bands in the gels were photographed using a Gel Doc XRS system (Bio-Rad, USA) and quantified by Quantity One software (Bio-Rad, USA).

2.7.

Data analysis

Values are expressed as the means ± SEM. Data were evaluated by one-way ANOVA followed by Dunnett’s or Fisher’s Protected Least Significant Difference test in SPSS 13.0 (SPSS, Chicago, IL, USA). Differences in p-values < 0.05 were considered significant.

3.

Results

3.1. Low-dose 3MC effects on the primary physiological parameters of mice No mortality was observed in any of the experimental groups throughout the 10 weeks of exposure to 3MC (0.01, 0.1 or 1 mg/kg BW/week). Body weights were higher in the HD groups than in the control group, indicating that feeding with an HD diet increased the body weights (Fig. 1A). Although no significant difference was observed between the HD and any one of the HD-3MC groups, the body weights in all 3MC-treated groups were higher than that of the HD group after 6 weeks of exposure. In comparison with the HD group, the body weights in the HD-0.1 and HD-1 groups increased by 7.6% and 6.2%, respectively, at the end of the exposure (Fig. 1A). The relative liver weight of the HD group was significantly higher than that of the BD group. However, no significant difference was observed between any of the 3MC-HD groups and the HD group (Fig. 1B). The relative fat weights in the HD and HD-0.1 groups were higher than that of the BD group (Fig. 1C). No significant difference was observed between the HD and any of the 3MC-treated groups (Fig. 1C). We observed that the sub-chronic exposure to 0.01, 0.1 and 1 mg/kg/week 3MC had no significant effects on the serum TG, TC, FFA or VLDL-C levels (Table 1). However, the FFA levels in the HD-1 group increased by approximately 12% when compared with that of the HD control group (Table 1).

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Fig. 1 – Sub-chronic 3MC exposure effects on mouse body weights, relative liver and fat weights. Adult male mice (7 weeks old) received intraperitoneal injections of 0, 0.01, 0.1 or 1 mg/kg BW 3MC per week for 10 weeks. After 10 weeks exposure, all the mice were sacrificed at the 7th day after the last time of intraperitoneal injection of 3MC. (A) Body weight during the exposure. (B, C) Relative liver and fat weights. The presented values are the means ± SE (n = 6). Different letters above the points indicate a significant difference (p < 0.05) between different groups, whereas the same letter indicates no significant difference.

3.2.

Hepatic histopathological changes

Representative histological liver sections stained with Oil red O (left) or H&E (right) at low and high magnifications are shown in Fig. 2A and B. In the hepatic histology assessed by Oil red O

staining, lipid droplets were barely observed in the BD group, but they increased significantly in the 3MC-treated HD groups (Fig. 2A). The H&E stain also indicated that many lipid vacuoles (as indicated by the arrow) were within the hepatocytes, especially in groups HD-0.1 and HD-1 (Fig. 2B). In addition, no

Table 1 – Sub-chronic 3MC exposure effects on blood levels of TG, FFA, TC, and VLDL-C in the serum.a Group HD HD-0.01 HD-0.1 HD-1 a

TG (mmol/L) 0.98 0.95 1.12 0.92

± ± ± ±

0.06 0.09 0.09 0.05

FFA (␮mol/L) 702.51 710.87 726.40 808.84

± ± ± ±

35.92 16.47 71.97 36.74

TC (mmol/L) 4.83 4.43 4.90 4.62

± ± ± ±

0.28 0.17 0.35 0.31

VLDL-C (mmol/L) 0.42 0.22 0.34 0.29

± ± ± ±

0.05 0.06 0.12 0.10

Adult male mice (7 weeks old) received intraperitoneal injections of 0, 0.01, 0.1 or 1 mg/kg BW 3MC per week for 10 weeks. After 10 weeks exposure, all the mice were sacrificed at the 7th day after the last time of intraperitoneal injection of 3MC. Values are presented as the means ± SE (n = 6). No significant difference was observed among any of the 3MC-treated groups and the HD group for all parameters.

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Fig. 2 – Sub-chronic 3MC exposure effects on mouse hepatic lipid accumulation. Adult male mice (7 weeks old) received intraperitoneal injections of 0, 0.01, 0.1 or 1 mg/kg BW 3MC per week for 10 weeks. After 10 weeks exposure, all the mice were sacrificed at the 7th day after the last time of intraperitoneal injection of 3MC. (A, B) Representative pictures and histochemistry of the liver. (A) Left panels, results of Oil-red O staining (B) Right panels, results of H&E staining. (C, D) Effect on triglyceride and cholesterol levels in the liver. The presented values are the means ± SE (n = 6). Different letters above the adjacent bars indicate a significant difference (p < 0.05) among different groups, whereas the same letter indicates no significant difference.

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serious cell damage was observed in the livers of 3MC-treated groups (Fig. 2B). Taken together, these histological examinations highlighted the development of lipid accumulation and confirmed that 3MC significantly affected lipid metabolism. Next, we further determined the TG and TCH levels in the liver. We observed that the hepatic TG and TCH levels in 3MC-treated HD groups were significantly higher than those in the HD group (Fig. 2C and D). The TG levels in the livers increased by approximately 1.4-, 1.5- and 2.1-fold in the HD-0.01, HD-0.1 and HD-1 groups, respectively, compared with that of the HD group (Fig. 2C). The hepatic TCH levels in the HD-0.1 and HD-1 groups were also approximately 1.3- and 1.4-fold higher than those in the corresponding HD groups, respectively (Fig. 2D).

3.3. 3MC effects on the expression of transcription factors related to fat metabolism in the liver and adipose tissue The Ppar mRNA levels increased by 1.51-, 1.53- and 2.69-fold in the livers of male mice following exposure to 0.01, 0.1 and 1 mg/kg BW/week 3MC, respectively, compared with that of the HD group (Fig. 3A). The Srebp1c transcriptional levels decreased significantly in the HD-0.1 and HD-1 groups when compared with that of the HD group (Fig. 3A). In contrast, the hepatic Ppar˛ mRNA levels were not influenced by the sub-chronic 3MC treatment. The Ppar mRNA levels increased in a dose-dependent manner in the adipose tissue of 3MC-treated groups, and they were approximately 3.2- and 5.4-fold higher in the HD-0.1 and HD-1 groups than those of the HD group, respectively (Fig. 3B). However, the Srebp1c mRNA levels also increased in the adipose tissue after the sub-chronic treatment with 3MC, and a significant difference was observed between the HD and HD-1 groups (Fig. 3B). According to the results of Western blot, the molecular weights of all the interest proteins were all as expected. In accordance with the mRNA levels, the Western blot results demonstrated that the PPAR protein levels in both the liver and adipose tissue also increased in a dose-dependent manner in the HD-0.01, HD-0.1 and HD-1 groups (Fig. 3C and D). The SREBP1 protein levels in the liver and adipose tissue increased, especially in the HD-0.1 and HD-1 groups (Fig. 3C and D). In accordance with the transcriptional status, the PPAR˛ protein levels in the liver were not affected by the sub-chronic exposure to 3MC (Fig. 3C).

3.4. 3MC effects on the transcription of genes related to fatty acid synthesis, oxidation and transport and TG synthesis in the liver We observed that the sub-chronic 3MC exposure tended to decrease the liver mRNA levels of the primary genes related to fatty acid synthesis including Acc, Fas and Scd1 (Fig. 4A). The Acc mRNA levels in the HD-1 group were significantly lower than that of the HD group, and the Fas mRNA levels in the HD-0.1 and HD-1 groups were also lower than that in the HD group. Moreover, the Scd1 mRNA levels decreased significantly in all 3MC-treated groups in comparison with that of the HD group. These results indicated that the de novo fatty acid

Fig. 3 – Sub-chronic 3MC exposure effects on the expression of genes (A and B) and proteins (C and D) of PPAR␣ PPAR␥ and SREBP1 in the livers and epididymal fat tissue of mice. Adult male mice (7 weeks old) received intraperitoneal injections of 0, 0.01, 0.1 or 1 mg/kg BW 3MC per week for 10 weeks. After 10 weeks exposure, all the mice were sacrificed at the 7th day after the last time of intraperitoneal injection of 3MC. The mice were fed the HD during the whole treatment period. The presented values are the means ± SE (n = 6). Different letters above the adjacent bars indicate a significant difference (p < 0.05) between groups, whereas the same letter indicates no significant difference.

synthesis capacity of the liver was decreased when the mice were sub-chronically exposed to 3MC. In addition, the mRNA levels of the primary genes related to fatty oxidation, Cpt1, Acox and Hsl in the livers of male mice were not affected by the sub-chronic 3MC exposure (Fig. 4B). FAT and FATP2 are primarily responsible for transferring FFA from the blood into the hepatocytes. We observed that the sub-chronic 3MC exposure increased Fat and Fatp2 transcription in the liver (Fig. 4C). The Fat transcript levels increased significantly in the HD-0.01 and HD-1 groups in comparison with those of the HD group (Fig. 4C). In addition, the Fatp2

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Fig. 5 – Sub-chronic 3MC exposure effects on histopathological changes (A) and the transcription of genes related to fatty acid synthesis (B) and TG hydrolysis (C) in mouse epididymal fat tissue. Adult male mice (7 weeks old) received intraperitoneal injections of 0, 0.01, 0.1 or 1 mg/kg BW 3MC per week for 10 weeks. After 10 weeks exposure, all the mice were sacrificed at the 7th day after the last time of intraperitoneal injection of 3MC. The mice were fed HD during the whole treatment period. The presented values are the means ± SE (n = 6). Different letters above the adjacent bars indicate a significant difference (p < 0.05) between different groups, whereas the same letter indicates no significant difference.

Fig. 4 – Sub-chronic 3MC exposure effects on the transcription of genes related to fatty acid synthesis (A), fatty acid oxidation (B), fatty acid transport (C) and TG synthesis (D) in the mouse liver. Adult male mice (7 weeks old) received intraperitoneal injections of 0, 0.01, 0.1 or 1 mg/kg BW 3MC per week for 10 weeks. After 10 weeks exposure, all the mice were sacrificed at the 7th day after the last time of intraperitoneal injection of 3MC. The mice were given HD during the whole treatment period. The presented values are the means ± SE (n = 6). Different letters above the adjacent bars indicate a significant difference (p < 0.05) between different groups, whereas the same letter indicates no significant difference.

mRNA levels in the HD-1 group were also significantly higher than those in the HD group (Fig. 4C). Regarding the TG synthesis genes, the hepatic Dgat2 mRNA levels were not influenced by the sub-chronic 3MC exposure (Fig. 4D) while the Gpat transcription decreased in a dose-dependent manner. A significant decrease was observed between the HD and HD-1 groups (Fig. 4D).

3.5. 3MC effects on histopathological changes and the transcription of genes related to fatty acid synthesis and TG hydrolysis in adipose tissue Representative histological sections of epididymal fat tissue were stained with H&E, and they indicated that the cell sizes were not significantly influenced by the sub-chronic 3MC exposure under a high-fat diet (Fig. 5A). However, the mRNA

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levels of the primary genes related to fatty acid synthesis including Acc, Fas and Scd1 increased in a dose-dependent manner in the epididymal fat (Fig. 5B). The Acc mRNA levels in the HD-0.1 and HD-1 groups were significantly higher than that of the HD group. The Fas mRNA levels increased by 2.5-, 2.8 and 2.9-fold in the HD-0.01, HD-0.1 and HD-1 groups, respectively, compared with that of the HD group. In relation to Scd1, the mRNA levels increased by 2.1-, 5.3- and 7.3-fold in the HD-0.01, HD-0.1 and HD-1 groups when compared with that of the HD group, respectively. In addition, the transcriptional levels of Hsl, which mainly catalyzes the rate-limiting step in stored TG hydrolysis in adipose tissue, also increased significantly, especially in the HD-1 group (Fig. 5C). These results indicated that the fatty acid levels in the adipose tissue were enhanced by synthesis and lipid metabolism when the mice were sub-chronically exposed to 3MC for 10 weeks.

4.

Discussion

Because of the widespread distribution of PAHs in the environment, it is likely that human and wildlife exposure to PAHs and their metabolites will increase around the world. PAHs including 3MC are agonists of AHR, which plays very important roles in the homeostatic control of lipid metabolism in the liver (Tanos et al., 2012). AHR gene and protein levels increased in both in vivo and in vitro systems following an acute exposure to 3MC for a short time (Kawano et al., 2010; Ovesen et al., 2011; Jin et al., 2013). However, in the present study, we observed that the mRNA and protein levels did not significantly increase on the 7th day after injection of the indicated 3MC dosage (data not shown). We further confirmed that the expression level of the primary cyp1a1 target gene in the liver was significantly induced by a 1 mg/kg 3MC injection after 1 day, but it returned to basal levels after 3 and 7 days (Fig. S1), indicating that the AHR was activated within 24 h after the low-dose injection. Thus, a possible explanation is that very low doses (0.01, 0.1 and 1 mg/kg/week) of 3MC were administered or 3MC was quickly metabolized and eliminated from the liver. Moorthy (2000) demonstrated that only 0.94% and 0.3% of the radioactivity of the administered dose remained in the liver after 1 and 8 days, respectively, when the rats received an intraperitoneal injection of 93 mol/kg [3H]MC once daily for 4 days. Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.etap. 2014.07.014. A number of previous studies demonstrated that some AHR agonists such as TCDD can induce hepatic lipid metabolism and steatosis under chronic toxicity (Boverhof et al., 2006). However, Kawano et al. (2010) also reported that a single intraperitoneal 3MC injection at 100 mg/kg body weight (BW) induced hepatic steatosis after 8 h. The present study is the first hepatic toxicity evaluation of the sub-chronic exposure to a PAH, namely 3MC. Low doses and long periods of exposure to 3MC slightly increased mouse body weights (Fig. 1); however, 0.1 and 1 mg/kg/week 3MC exposure impaired glucose tolerance at the sixth week as monitored by the glucose tolerance test (Fig. S2). We also observed that the fat weights (Fig. 1C) and cell sizes (Fig. 5A) were not influenced by 3MC treatment.

Moreover, the most interesting findings showed that lipid accumulation increased significantly in the liver (Fig. 2A and B) and the hepatic levels of TG and TC increased significantly in the HD-0.1 and HD-1 groups (Fig. 2C and D). These results directly suggested that the sub-chronic 3MC exposure mainly influenced the lipid metabolism of the liver. Supplementary Fig. S2 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.etap. 2014.07.014. As reported, PPAR˛/ and SREBP1C are the primary nuclear transcriptional factors involved in mammalian fat metabolism (Wahli et al., 1995; Schoonjans et al., 1996; Nguyen et al., 2008; Gao et al., 2013). In the present study, we observed that the sub-chronic 3MC exposure disturbed the transcriptional and translational status of PPAR and SREBP1C in the liver and adipose tissue of mice when a high-fat diet was provided (Fig. 3). PPARs play a role in improving several perturbations of metabolic syndrome (Medjakovic et al., 2010). The primary functions of PPAR are adipocyte differentiation and insulin sensitization (Gregoire et al., 1998). PPAR can also selectively up-regulate a subset of the lipogenic enzymes in hepatocytes, thereby enhancing lipid synthesis in the liver (Coleman and Lee, 2004). PPAR˛ is highly expressed in the liver and in tissues in which large amounts of lipid-derived energy are used, and PPAR˛ regulates a set of enzymes that are crucial for fatty acid oxidation (Nguyen et al., 2008). In fat metabolism, SREBP1C is mainly responsible for the expression of downstream genes, such as Fas, Scd1 and Acc, which are related to fatty acid synthesis (Hagiwara et al., 2012). Some previous studies showed that the AHR regulated the expression of these factors in a different manner. For example, Wang et al. (2011) reported that ˇ-naphthoflavone, an agonist of AHR, stimulated PPAR˛ expression in WT but not in AHR KO mice. Moreover, AHR antagonist ˛-naphthoflavone blocks the ˇ-naphthoflavone induction of PPAR˛ indicating that PPAR˛ is regulated by AHR. Tanos et al. (2012) demonstrated a downregulation of the SREBP1C target genes in AHR ligand-treated mice and primary human hepatocytes and their increased expression in the absence of AHR, suggesting that a possible interaction existed between AHR and SREBP1C. In our experiment, the PPAR and SREBP1C expressions were significantly altered in the liver and adipose tissue following the sub-chronic 3MC exposure. When combining this information with previous reports, it is possible that the lipid accumulation in the liver caused by the sub-chronic exposure to 3MC is closely related to the status of transcription factors such as PPAR and SRCBP1C. However, the primary mechanisms underlying the link between AHR and these transcription factors in relation to fat metabolism have remained unclear. At the molecular level, lipid metabolism is controlled by a series of key genes that are involved in fatty acid synthesis, oxidation and transport and TG synthesis in the liver (Nguyen et al., 2008; Palou et al., 2008). In the present study, we found that the sub-chronic 3MC exposure decreased the transcription of genes involved in fatty acid synthesis in the liver in a dose-dependent manner (Fig. 5A), indicating that de novo fatty acid synthesis in hepatocytes was significantly inhibited by the sub-chronic 3MC exposure. Similarly, Tanos et al. (2012) reported that the normalized RNA expression of hepatic fatty acid synthesis genes including Acc, Fas, and Scd1

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Fig. 6 – Summary of the possible mechanisms underlying the influence of 3MC exposure on hepatic lipid accumulation in mice. Sub-chronic exposure to 3MC activated the AHR, which negatively regulated the synthesis in the liver. The mRNA levels of genes related to FFA synthesis such as Acc, Fas and Scd1 decreased significantly, indicating that the de novo fatty acid synthesis in hepatocytes was significantly inhibited by sub-chronic 3MC exposure. In contrast, sub-chronic exposed to 3MC greatly enhanced the FFA synthesis greatly mediated by up-regulation the expression of PPAR␥, SREBP1C and target genes including Acc, Fas and Scd1 in the adipose tissue. FFA may be released into the blood and transported into the liver through the up-regulation of Fat and Fatp2, which resulted in the gradual accumulation of lipids in the liver.

decreased significantly in C57BL/6J mice when injected with another AHR agonist, namely ˇ-naphthoflavone, at 50 mg/kg for 5 h when compared to control mice. In contrast, the expression of these genes did not change in AHR-deficient mice under the same exposure conditions. Considering these findings, the AHR negatively regulated the expression of hepatic fatty acid synthesis genes in mice. However, the mRNA levels of Cpt1, Acox and Hsl were not affected in the liver (Fig. 4B), indicating that the fatty acid oxidation process was not influenced by the sub-chronic 3MC exposure. Moreover, the Gpat mRNA levels decreased in a dose-dependent manner. Because the hepatic TG contents in the 3MC-treated mice were significantly higher than that of the HD control, the decreased Gpat expression might have been caused by negative feedback inhibition. In addition, the serum VLDL levels did not increase significantly (Table 1), suggesting that hepatic TG was not transferred into the blood. Interestingly, Fat and Fatp2 significantly increased among the primary genes related to fatty acid transport, especially in the HD-1 group (Fig. 4C). As the integral trans-membrane proteins, FAT and FATP2 mostly mediated cellular uptake of very long-chain and long chain fatty acids (Mishima et al., 2011). Thus, our results suggested that the sub-chronic 3MC exposure enhanced FFA transport from the blood into the liver. Similarly, Kawano et al. (2010) reported that acute exposure to 100 mg/kg 3MC for 8 h induced hepatic steatosis via the up-regulation of fatty acid transport. Because the serum FFA did not change significantly following

the sub-chronic 3MC exposure (Table 1), we further determined the transcriptional levels of the genes related to FFA synthesis in the adipose tissue to identify the origin of FFA. We observed that both the up-stream transcriptional factors including PPAR and SREBP1C and the down-stream genes including Fas, Scd1 and Acc significantly increased after the sub-chronic 3MC exposure (Figs. 4B and 5B). Additionally, the Hsl transcriptional levels also increased (Fig. 5C). In adipose tissue, HSL is mainly responsible for catalyzing the rate-limiting step in the hydrolysis of stored TG (Fredrikson et al., 1981). These results suggested that the FFA synthesis increased in adipose tissue when the mice were sub-chronically exposed to 3MC. More importantly, because the adipose tissue weights (Fig. 1C) and cell sizes (Fig. 5A) did not increase to a corresponding degree, the synthesized FFA could have been released into the blood. Thus, it is possible that these FFAs were transported into the liver by FAT and FATP2, resulting in lipid accumulation in the livers.

5.

Conclusions

Our results indicate hepatic lipid accumulation is a consequence of sub-chronic PAH exposure, and it is characterized by a disturbance in the gene transcriptional status in relation to the liver lipid metabolism of male mice. These results suggest that the sub-chronic PAH exposure plays an

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important role in the development of hepatic lipid accumulation as induced by a high-fat diet through the regulation of lipid metabolism-related gene expression. Potential mechanisms through which the sub-chronic 3MC exposure might induce lipid accumulation in the liver are depicted in Fig. 6. In brief, the sub-chronic 3MC exposure activated the AHR, which negatively regulated the synthesis of fat in the liver. The primary genes including Acc, Fas and Scd1 decreased significantly compared with those in the control group, indicating that the de novo fatty acid synthesis in hepatocytes was significantly inhibited by the sub-chronic 3MC exposure. In contrast, the sub-chronic 3MC exposure greatly enhanced FFA synthesis in the adipose tissue by up-regulating the expression of PPAR, SREBP1C and their target genes including Acc, Fas and Scd1. The synthesized FFA was released into the blood and then transported into the liver by the up-regulation of Fat and Fatp2, which resulted in the gradual accumulation of lipids in the liver. On the basis of evidence presented in this study, we conclude that environmental PAHs might be an important contributing factor in the development of hepatic steatosis and hepatotoxicity in mammals. In fact, humans and wildlife are exposed to extremely low concentrations of different PAHs throughout most of their life spans. Thus, it is clear that we must perform further research on the specific contribution of PAH exposure to lipid metabolism and the obesity epidemic.

Conflict of interest The authors declare that there are no conflicts of interest.

Transparency document The Transparency document associated with this article can be found in the online version.

Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (nos. 21277128; 21107098) and the National Basic Research Program of China (no. 2010CB126100).

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Sub-chronically exposing mice to a polycyclic aromatic hydrocarbon increases lipid accumulation in their livers.

The potential for exposing humans and wildlife to environmental polycyclic aromatic hydrocarbons (PAHs) has increased. Risk assessments describing how...
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