Activation of sterol regulatory element-binding proteins in mice

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1 Activation of sterol regulatory element-binding proteins in mice exposed to perfluorooctanoic acid for 28 days Shengmin Yan, Jianshe Wang, and Jiayin Dai* Materials and methods Chemicals and antibodies Perfluorooctanoic acid (PFOA, CAS number , 96% purity) and WY-14,643 (CAS number , 98% purity) were obtained from Sigma-Aldrich (St. Louis, MO). Anti-SREBP1 mouse monoclonal antibody, anti-srebp2 rabbit polyclonal antibody, anti FBXW7 rabbit monoclonal antibody, and anti INSIG2 rabbit polyclonal were purchased from Abcam (Cambridge, MA). Anti-INSIG1 goat polyclonal antibody, anti-scap goat polyclonal antibody, HRP-conjugated goat anti-mouse, HRP-conjugated goat anti-rabbit, and HRP-conjugated donkey anti-goat were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-tubulin mouse monoclonal antibody was purchased from Beijing CoWin Bioscience Co, Ltd (Beijing, China). All other chemicals used were of the highest grade commercially available. Animal treatment Male Balb/c mice (age 6-8 weeks) were obtained from Weitong Lihua Experimental Animal Center (Beijing, China) and all experimental manipulations were described as our previous study (Yan et al. 2014). In brief, mice were randomly divided into six groups and dosed with either Milli-Q water or PFOA diluted in Milli-Q water at doses of 0.08, 0.31, 1.25, 5 and 20 mg/kg/d by gavage 1

2 for 28 days. The doses of PFOA were chosen according to earlier studies and our previous experiments. All animal treatment were approved by the Committee on the Ethics of Animal Experiments from the Institute of Zoology, Chinese Academy of Sciences (Permit Number: EET ). Liver PFOA determination Liver PFOA concentrations of each group (n = 3) were analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Liver samples were weighted and prepared as homogenates with Milli-Q water for PFOA analysis. PFOA was extracted as our previous report with some modification (Zhang et al. 2012). Briefly, about 100 mg liver samples were extracted with acetonitrile (ACN) twice, with both extractants then combined and concentrated to nearly 0.5 ml under a nitrogen gas flow at 40 C. Each concentrated solution were added with 0.5 ml of methanol (MeOH) then diluted using 10 ml of Milli-Q water for SPE cleanup. We passed each solution through pre-equilibrated Oasis WAX cartridges (Oasis HLB; 150 mg, 6cc; Waters). The cartridges were first rinsed with Milli-Q water and then 25 mm of acetate buffer solution (ph 4) after loading all samples. Remaining water in the cartridges was removed using vacuum filtration. PFOA were eluted using 4 ml of MeOH and 4 ml of 0.1% NH 4 OH in MeOH then concentrated to approximately 1 ml under nitrogen gas. PFOA determination and data analysis were performed as our previous report (Yan et al. 2014). Cell culture The mouse hepatocellular carcinoma cell line Hepa 1-6 (Cell Resource Center, IBMS, CAMS/PUMC) was cultured in DMEM supplemented with 2

3 10 % fetal bovine serum (FBS) in 25-cm 2 tissue culture flasks at 37 C in a humidified atmosphere composed of 95 % air and 5 % CO 2. PFOA was dissolved as a stock solution of 10 mm in serum free DMEM and then filter sterilized (0.22 μm Millipore filter, Millipore, USA). WY-14,643 was dissolved in absolute ethanol as a stock solution of 10 mm. All stock solutions were diluted with DMEM supplemented with 10 % FBS before use. According to our Hepa 1-6 cell viability assessment by MTT test, we found PFOA did not reduce cell viability below the dose of 250 μm (Fig.S1) after 72 h treatment and doses of 50, 100 and 200 μm were chosen for determine toxic effects of PFOA on Hepa 1-6 cells. According to earlier toxicological study(takacs and Abbott 2007), WY-14,643 at the dose of 25 μm was used as a positive control for PPARα activation and 1/400 (V/V) ethanol (cell viability of Hepa1-6 after exposure to these two chemicals around the chosen concentration for 72 h was tested and shown in Fig. S2) were used as its solvent control. For total RNAs isolation, protein isolation, and determination of total cholesterol (TCHO) and triglyceride (TG) concentrations, cells were plated at a density of cells per well in 6-well tissue culture plates containing 2 ml of culture medium per well. The medium was replaced with PFOA, ethanol or WY-14,643 at the doses mentioned above overnight after plating and exposed for 72 h before later use. For flow cytometry, cells were plated at a density of cells per well in 12-well tissue culture plates containing 1 ml of culture medium per well and treated as mentioned above. 3

4 Determination of TCHO and TG concentrations Tissue total cholesterol assay kit and tissue triglyceride assay kit (Applygen Technologies, Beijing, China) were used for determination of TCHO and TG concentrations. Mouse liver homogenates or Hepa 1-6 cell lysates were prepared and TCHO and TG concentrations were determined according to the manufacturer s instructions. bicinchoninic acid (BCA) protein assay kit (Tiangen, Beijing, China) was used detect protein concentrations in the tissue homogenates or cell lysates and the concentrations of TCHO or TG were then adjusted by protein concentrations. Detection of neutral lipids change in cells Neutral lipids change in Hepa 1-6 cell after exposed to PFOA were detected by flow cytometry using the fluorescent neutral lipid dye BODIPY 493/503 (Molecular Probes, Carlsbad, CA). BODIPY 493/503 were prepared as a stock solution of 10 mg/ml in ethanol (Gocze and Freeman 1994) and diluted to 0.2 μg/ml in PBS as working solution before use. After exposed to PFOA for 72 h, cells were harvested by trypsinization, washed twice with PBS, and then incubated with 0.2 μg/ml BODIPY 493/503 for 30 min in the dark. Samples were then processed on a BD FACS Calibur with Cell Quest software (BD Bioscience, San Jose, CA) with the preset yellow channel that detected emitted light maximally at 520 nm. At least 20,000 cells in each sample were analyzed and the mean fluorescence intensity was used to calculate the fold change of neutral lipids contents. Real-time PCR analysis Total RNAs were isolated from mouse livers (n = 6) or Hepa 1-6 cells using TRIzol (Life Technologies-Invitrogen, Carlsbad, CA, USA) 4

5 according to the manufacturer s instructions and measured by a NanoDrop 2000 spectrophotometer (Thermo Scientific). cdnas were synthesized using an oligo-(dt) 15 primer and M-MLV reverse transcriptase (Promega, Madison, USA). The messenger RNA (mrna) expression levels of selected genes were quantified through real-time PCRs performed with the Stratagene Mx3000P q-pcr system (Stratagene, USA) using SYBR Green PCR Master Mix reagent kits (Tiangen, Beijing, China) according to the manufacturer s instructions. Mouse specific primers were designed for selected genes (Table S1 in supplementary Information). Data were analysis with MxPro QPCR software and the relative quantification of each mirna was calculated using the 2 ΔΔCt method using 18S ribosomal RNA (18S) as the endogenous control. Protein isolation Frozen liver specimens were homogenized in RIPA buffer (Thermo Scientific) containing 1 mm phenylmethanesulfonyl fluoride (PMSF) and then collect the supernatant after centrifuged at g for 15 min. After treated for 72 h, Hepa 1-6 cells were washed with ice cold PBS and lysed with RIPA buffer containing 1 mm PMSF. The cell lysates were collected and centrifuged at g for 15 min. Supernatant was transferred to a new tube for further analysis. Protein concentration was determined using BCA protein assay kit (Tiangen, Beijing, China). Western blotting The proteins (40 μg of tissue protein or 15 μg of cell protein) were separated using a 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, Massachusetts), which were then blocked for 1 h at room temperature using 5 % bovine serum albumin (BSA) in Tris-buffered 5

6 saline containing 0.1 % Tween 20 (TBST). Membranes were incubated with the appropriate primary antibody diluted with 5 % BSA in TBST containing 0.02 % NaN 3 at 4 C overnight and then washed with TBST before incubated with horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) diluted with 5 % non-fat milk or BSA in TBST at room temperature. Protein bands were detected using enhanced chemiluminescence (ECL) system Western blot detection kit (Tiangen, Beijing, China) and were visualized by exposed to X-ray film (Kodak, NY). Membranes were subsequently washed and then reprobed for β-tubulin to equal protein loading. The intensities of protein bands were quantified using Quantity One software (Bio-Rad, Hercules, CA). TaqMan mirna assay About 50 ng RNA from mouse liver or Hepa 1-6 cells were used for TaqMan mirna assay as mentioned in our previous report (Yan et al. 2014). The mirnas analyzed in this study were mmu-mir-96-5p (assay ID ), mmu-mir-182-5p (assay ID ), mmu-mir-183-5p (assay ID ). U6 snrna (assay ID ) was selected as the endogenous control, and fold change of each mirna after exposed to PFOA was calculated using the 2 ΔΔCt method. Statistical analysis Statistical analyses were performed using SPSS for Windows 17.0 Software (SPSS, Inc., Chicago, IL). Data were represented as means with standard errors (mean ± S.E.). Differences between treatments were determined using one-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) test, with p-value of < 0.05 deemed significant. All represented 6

7 mean values ± S.E. from cell experiments were based on at least 3 independent experiments. References Gocze PM, Freeman DA (1994) Factors underlying the variability of lipid droplet fluorescence in MA-10 Leydig tumor cells. Cytometry 17(2):151-8 Takacs ML, Abbott BD (2007) Activation of mouse and human peroxisome proliferator-activated receptors (alpha, beta/delta, gamma) by perfluorooctanoic acid and perfluorooctane sulfonate. Toxicol Sci 95(1): Yan S, Wang J, Zhang W, Dai J (2014) Circulating MicroRNA Profiles Altered in Mice after 28 Days Exposure to Perfluorooctanoic Acid. Toxicol Lett 224(1):24-31 Zhang W, Zhang Y, Zhang H, Wang J, Cui R, Dai J (2012) Sex differences in transcriptional expression of FABPs in zebrafish liver after chronic perfluorononanoic acid exposure. Environ Sci Technol 46(9):

8 Viability (%) h Dose of PFOA ( M) Fig. S1 Hepa 1-6 cells viability after exposure to PFOA for 72 hours, Data are means ± SE from each of the three experiments was performed in quadruply. 8

9 Cell viability (%) * * 0 1/1000 1/400 1/ Ethanol (V/V) WY ( M) Fig. S2 Hepa 1-6 cells viability after exposed to Ethanol or WY-14,643 (WY) for 48 and 72 hours, Data are means ± SE from each of the three experiments was performed in quadruply, significantly different from control group (*p < 0.05, **p < 0.01). 9

10 Relative mrna level mg/kg/d 0.08 mg/kg/d 0.31 mg/kg/d 1.25 mg/kg/d 5 mg/kg/d 20 mg/kg/d 1 0 ** ** Hnf4 Aldob Cyp1a2 Cyp7a1 L-pk Pck1 Fig. S3 Relative mrna level of Hnf-4α and its representative target genes (n = 6). Data are means ± SE. Significantly different from control group (*p < 0.05, **p < 0.01). 10

11 Relative mrna level mg/kg/d 0.08 mg/kg/d 0.31 mg/kg/d 1.25 mg/kg/d 5 mg/kg/d 20 mg/kg/d ** ** * 1 0 Fbxw7 Insig1 Insig2 * Scap Fig. S4 Relative fold change of Fbxw7, Insig1, Insig2 and Scap mrna level (n = 6). Data are means ± SE. Significantly different from control group (*p < 0.05, **p < 0.01). 11

12 Fig. S5 A possible schematic model of how SREBP maturation pathway involved in the effects induced by PFOA on liver metabolism. 12

13 13

14 Table S1 Primers for qpcr MOUSE NM (bp) Forward (5 to 3 ) Reverse (5 to 3 ) 18S NR_ GCCGTTCTTAGTTGGTGGA GACCTGTTATTGCTCAATCTCG Acox1 NM_ TAACTTCCTCACTCGAAGCCA AGTTCCATGACCCATCTCTGTC Aldob NM_ GAAACCGCCTGCAAAGGATAA GAGGGTCTCGTGGAAAAGGAT Apoa4 NM_ GGAGCACCTGAAGCCCTAT ATGCGCTGGATGTATGG Apoc3 NM_ AAGTAGCGTGCAGGAGTC ATAGCTGGAGTTGGTTGG Cpt1a NM_ TGGCATCATCACTGGTGTGTT GTCTAGGGTCCGATTGATCTTTG Cyp1a2 NM_ AGTACATCTCCTTAGCCCCAG GGTCCGGGTGGATTCTTCAG Cyp4a10 NM_ CCCAAGTGCCTTTCCTA GCAAACCATACCCAATCC Cyp7a1 NM_ AATCTACCCAGACCCTT TTGCTTTCCCACTTTC Dbi (Acbp) NM_ GAATTTGACAAAGCCGCTGAG CCCACAGTAGCTTGTTTGAAGTG Fabp1(L-fabp) NM_ ATGAACTTCTCCGGCAAGTACC CTGACACCCCCTTGATGTCC Fasn NM_ GGCTCTATGGATTACCCAAGC CCAGTGTTCGTTCCTCGGA Fbxw7 NM_ GGCACTCTATGTGCTTTCATTCC TGTCTGATATACGCACTCTTCCA Hmgcr NM_ GATTCTGGCAGTCAGTGGGAA GTTGTAGCCGCCTATGCTCC Hmgcs1 NM_ AACTGGTGCAGAAATCTCTAGC GGTTGAATAGCTCAGAACTAGCC Hmgcs2 NM_ GAAGAGAGCGATGCAGGAAAC GTCCACATATTGGGCTGGAAA Hnf4α NM_ AAGGTGCCAACCTCAATTCATC CACATTGTCGGCTAAACCTGC Insig1 NM_ TGTCGGTTTACTGTATCCCTGT GTTGATGCCAACGAACACGG Insig2 NM_ GGAGTCACCTCGGCCTAAAAA CAAGTTCAACACTAATGCCAGGA Ldlr NM_ GTCTGTCACCTGTCAGTCCAATC TCTAGGCAATCTCGGTCTCC Lpl NM_ TCTCCTGATGACGCTGAT CTTGGAGTTGCACCTGTAT Pck1(Pepck) NM_ CTGCATAACGGTCTGGACTTC CAGCAACTGCCCGTACTCC Pklr(L-pk) NM_ TCAAGGCAGGGATGAACATTG CACGGGTCTGTAGCTGAGTG Ppar-a NM_ AGAGGTCCGATTCTTCCA ATCCAGTTCTAAGGCATTGA Ppar-d NM_ GCAGCCTCAACATGGAATGTC GAGCTTCATGCGGATTGTCC Ppar-g NM_ GGAAGACCACTCGCATTCCTT GTAATCAGCAACCATTGGGTCA Scap NM_ CTGACGCCCACACTCAATG CACCAACACGTTCTCTAGCCC Scd1 NM_ GACCTGAAAGCCGAGAA CGTTGAGCACCAGAGTGT Srebf1 NM_ CAAGGCCATCGACTACATCCG GCCCTCCATAGACACATCTGT Srebf2 NM_ GTGGGAGAGTTCCCTGATTTG CTCCACCATTGTTGCCTCTG Vldlr NM_ AGACCAATCAGACGAGTCTCTT CTGCCGTCCTTGCAGTCAG 14

15 Abbreviations PFAAs, perfluoroalkyl acids PFOA, perfluorooctanoic acid TCHO, total cholesterol TG, triglyceride 18S, 18S ribosomal RNA Acox1, acyl-coenzyme A oxidase 1, palmitoyl Aldob, aldolase B, fructose-bisphosphate Apoa4, apolipoprotein A-IV Apoc3, apolipoprotein C-III CAR, constitutive active receptor Cpt1a, carnitine palmitoyltransferase 1a, liver Cyp1a2, cytochrome P450, family 1, subfamily a, polypeptide 2 Cyp4a10, cytochrome P450, family 4, subfamily a, polypeptide 10 Cyp7a1, cytochrome P450, family 7, subfamily a, polypeptide 1 Dbi (Acbp), diazepam binding inhibitor Fabp1, fatty acid binding protein 1, liver Fasn, fatty acid synthase Fbxw7, F-box and WD-40 domain protein 7 FXR, farnesoid X receptor Hmgcr, 3-hydroxy-3-methylglutaryl-Coenzyme A reductase Hmgcs1, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 Hmgcs2, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 Hnf4a, hepatic nuclear factor 4, alpha Insig1, insulin induced gene 1 Insig2, insulin induced gene 2 Ldlr, low density lipoprotein receptor Lpl, lipoprotein lipase LXR, liver X receptor Pck1(Pepck), phosphoenolpyruvate carboxykinase 1, cytosolic Pklr(L-pk), pyruvate kinase liver and red blood cell Ppar-a, peroxisome proliferator activated receptor alpha Ppar-d, peroxisome proliferator activator receptor delta Ppar-g, peroxisome proliferator activated receptor gamma PXR, pregnane X receptor RXR, retinoid X receptors Scap, SREBF chaperone Scd1, stearoyl-coenzyme A desaturase 1 Srebf1, sterol regulatory element binding transcription factor 1 Srebf2, sterol regulatory element binding factor 2 Vldlr, very low density lipoprotein receptor 15