Arch Toxicol DOI 10.1007/s00204-014-1322-7 MOLECULAR TOXICOLOGY Activation of sterol regulatory element‑binding proteins in mice exposed to perfluorooctanoic acid for 28 days Shengmin Yan · Jianshe Wang · Jiayin Dai Received: 10 April 2014 / Accepted: 21 July 2014 © Springer-Verlag Berlin Heidelberg 2014 Abstract Perfluoroalkyl acids are widely used in numer- lipid content and PPARα in Hepa 1-6 cells after exposure ous industrial and commercial applications due to their to PFOA for 72 h but SREBPs were not activated in vitro. unique physical and chemical characteristics. Although per- These results demonstrated that SREBPs were maturated fluorooctanoic acid (PFOA) is associated with hepatomeg- by activating the miR-183-96-182 cluster-SREBP regula- aly through peroxisome proliferator-activated receptor α tory loop in PFOA-exposed mouse liver. (PPARα) activation, liver fat accumulation and changes in gene expression related to fatty acid metabolism could still Keywords SREBP · Lipid metabolism · PPAR · be found in PPARα-null mice exposed to PFOA. To explore Perfluorononanoic acid the potential effects of PFOA on sterol regulatory element- binding proteins (SREBPs) activity, male mice were dosed with either Milli-Q water or PFOA at doses of 0.08, 0.31, Introduction 1.25, 5, and 20 mg/kg/day by gavage for 28 days. Liver total cholesterol concentrations and PFOA contents showed Perfluoroalkyl acids (PFAAs) are a family of anthropo- a dose-dependent decrease and increase, respectively. Tran- genic compounds widely used in industrial and commer- scriptional activity of PPARα and SREBPs was signifi- cial applications due to their unique physical and chemical cantly enhanced in livers. Protein expression analyzed by characteristics (Lau et al. 2007). The toxicological effects Western blotting showed that PFOA exposure stimulated of two of the most widely known PFAAs, perfluorooctanoic SREBP maturation. Furthermore, proteins blocked SREBP acid (PFOA) and perfluorooctane sulfonate (PFOS), have precursor transport, insulin-induced gene 1 (INSIG1) and been extensively studied, and earlier reports have showed INSIG2 proteins, as well as a protein-mediated nuclear PFOA and PFOS to have similar effects in rodents, such SREBP proteolysis, F-box and WD-40 domain protein 7, as reduced body weight, increased liver weight, reduced decreased in mouse liver exposed to PFOA. The expres- serum cholesterol, and dose-dependent increased mortality sion levels of the miR-183-96-182 cluster, which is possi- (Lau et al. 2007). bly involved in a regulatory loop intermediated by SREBPs Among phenomena induced by PFOA and PFOS, liver maturation, were also increased in the mouse liver after enlargement in rodents is evident even at low-dose expo- PFOA exposure. We also observed that PFOA induced sure (Wan et al. 2012; Yan et al. 2014). Several studies have determined the reasons of PFAA-induced hepatomegaly and have shown that PFOA and PFOS are capable of induc- Electronic supplementary material The online version of this ing peroxisome proliferation, with activation of peroxisome article (doi:10.1007/s00204-014-1322-7) contains supplementary material, which is available to authorized users. proliferator-activated receptors (PPARs) the first key event (Lau et al. 2007; Takacs and Abbott 2007). PPARs are a S. Yan · J. Wang · J. Dai (*) group of closely related ligand-dependent nuclear recep- Key Laboratory of Animal Ecology and Conservation tors that mediate the effects of eicosanoids, fatty acids, and Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China synthetic peroxisome proliferators on gene transcription e-mail: [email protected] (Kersten et al. 2000). Three PPAR isotypes, peroxisome 1 3 Arch Toxicol proliferator-activated receptor α (PPARα), β/δ and γ, have 2013; Knight et al. 2005; Yoshikawa et al. 2003) in vivo and in been identified in various tissues encoded by separate genes vitro. However, the exact mechanism of the cross talk between and play specific roles in controlling lipid metabolism and PPARs and SREBPs is still unclear, especially the inconsist- inflammation (Kersten et al. 2000; Takacs and Abbott 2007; ent results in vitro treated with WY-14,643 or PPARα and γ Varga et al. 2011). Consistent with their master regulatory over expression. The induction of SREBP1c gene expression functions in metabolism, PPARs are highly expressed in at the transcriptional level by PFAAs has been reported before tissues most active in lipid metabolism, with PPARα highly (Bjork et al. 2011; Fang et al. 2012), but whether SREBPs expressed in the liver (Varga et al. 2011). PPARα plays an play a role in effects induced by PFAAs exposure on the liver important role in fatty acids β-oxidation, ketogenesis, and is still unclear. In the present study, we assessed the activation systemic lipid metabolism, and PPAR agonists reportedly of SREBPs, especially key factors in the maturation pathway induce hepatomegaly and hepatocarcinogenesis without of SREBPs, in vivo and in vitro after PFOA exposure. We genetic damage (Pyper et al. 2010). Although PFOA- and found that induction of the maturation pathway was involved PFOS-induced liver enlargement appears to result from in SREBPs activation after PFOA exposure. acting as a PPAR ligand, liver weight increase was still observed in PFOA-exposed PPARα-null mice (Minata et al. 2010; Wolf et al. 2008). Fat accumulation and Materials and methods changes in genes related to fatty acid metabolism were also observed in PFOA-treated PPARα-null mice (Minata et al. Animal treatment 2010; Rosen et al. 2008a, b), indicating that the activation of other alternate nuclear receptors may also be involved Male Balb/c mice (age 6–8 weeks) were obtained from in lipid metabolism disturbance induced by PFOA. Several the Weitong Lihua Experimental Animal Center (Beijing, potential targets of PFOA and PFOS toxicity, which may China), and all experimental manipulations are described in be independent or indirectly controlled via cross talk with our previous study (Yan et al. 2014). In brief, mice were PPARα, were previously analyzed in vitro and seem to be randomly divided into six groups and dosed with either activated in cells of rats more so than that of humans after Milli-Q water or PFOA diluted in Milli-Q water at doses exposure to PFOA or PFOS (Bjork et al. 2011). of 0.08, 0.31, 1.25, 5, and 20 mg/kg/day by gavage for The sterol regulatory element-binding proteins (SREBPs), 28 days. The doses of PFOA were chosen according to ear- a family of membrane-bound, basic helix-loop-helix leucine lier studies and our previous experiments. All animal treat- zipper (bHLH-LZ) transcription factors, play a critical role in ments were approved by the Committee on the Ethics of controlling cholesterol and lipid metabolism and are involved Animal Experiments from the Institute of Zoology, Chinese in several biological processes and disease states (Bengoe- Academy of Sciences (Permit Number: EET-015-08-2012). chea-Alonso and Ericsson 2007; Shao and Espenshade 2012). Three major SREBP proteins (SREBP1a, SREBP1c, Liver PFOA determination and SREBP2) are encoded by the Srebf1 and Srebf2 genes, and most data suggest that the regulatory functions of the PFOA was extracted from the livers of each group (n 3) = two SREBP1 isoforms are primarily in fatty acid metabolism and analyzed using high-performance liquid chromatogra- and SREBP2 is mainly in cholesterol metabolism (Jeon and phy–tandem mass spectrometry (HPLC–MS/MS). Osborne 2012; Shao and Espenshade 2012). Tissue distribu- tion of the three mammalian SREBPs differs, with SREBP1c Cell culture the predominant isoform in most adult nondividing meta- bolic tissues such as liver and adipose (Jeon and Osborne The mouse hepatocellular carcinoma cell line Hepa 1-6 2012; Raghow et al. 2008). SREBPs are translated as inac- (Cell Resource Center, IBMS, CAMS/PUMC) was used to tive precursors and activated by a set of elaborate maturation determine the effects of PFOA and WY-14,643 (a PPARα pathways, which are regulated by multiple signals. Sterol, ligand) in vitro. The doses of each chemical we chose insulin, and insulin-induced gene (INSIGs) play a key role were based on our previous cell viability test using the in sterol-dependent regulation of SREBP (Shao and Espen- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- shade 2012). Not only is the maturation pathway of SREBPs mide (MTT; Sigma–Aldrich, Steinheim, Germany) method adjusted under different conditions, nuclear SREBPs are also (Fig. S1 and S2). highly regulated by posttranslational modification, such as phosphorylation, acetylation, and ubiquitinylation (Bengoe- Determination of TCHO and TG concentrations chea-Alonso and Ericsson 2007). Earlier reports showed a complex cross talk between Tissue total cholesterol and tissue triglyceride assay kits PPARs and SREBP (Fernandez-Alvarez et al. 2011; Gao et al. (Applygen Technologies, Beijing, China) were used for 1 3 Arch Toxicol determination of liver total cholesterol (TCHO) and TG with p values of <0.05 deemed significant. All represented concentrations, respectively. mean SE from cell experiments were based on at least ± three independent experiments. Detection of neutral lipids change in cells Neutral lipid changes in Hepa 1-6 cells after exposure to Results PFOA were detected by flow cytometry using fluorescent neutral lipid dye BODIPY 493/503 (Molecular Probes, PFOA changed lipid content and activated PPARs in mouse Carlsbad, CA, USA). liver Real-time PCR analysis To investigate the effects of PFOA on hepatic lipid metabo- lism, mice were exposed to PFOA by gavage for 28 days, Total RNAs were isolated from the mice livers (n 6) and and liver TCHO and TG concentrations were then analyzed = Hepa 1-6 cells using TRIzol (Life Technologies-Invitro- (Fig. 1a, b). Results demonstrated that TCHO concentra- gen, Carlsbad, CA, USA) according to the manufacturer’s tions in the liver were reduced in a dose-dependent man- instructions and were measured by a NanoDrop 2000 spec- ner after exposure to PFOA, and TG concentrations were trophotometer (Thermo Scientific, Waltham, MA).