Chemicals and reagents
3MC, BaP, butylated hydroxylanisole (BHA), sulforaphane (SUL), menadione, tert-butylhydroquinone (t-BHQ), MDL28170, chloroquine, MG132, sulindac sulfide (SS) and sulindac sulfoxide (SOX) as well as antibodies to IgG used in the ChIP assay were obtained from Sigma-Aldrich (St. Louis, MO). Ethionamide sulfoxide (ETASO) was a kind gift from Dr. Paul R. Ortiz de Montellano (University of California, San Francisco, CA). Mouse anti-AHR antibody (SA-210) was from BIOMOL Research Laboratory (Plymouth Meeting, PA); human anti-p53 antibody (FL-393) and human anti-ARNT were from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). The Hepa-1c1c7 (Hepa-1) mouse hepatoma cell line, the AHR-deficient cell line c35 (B16GBi1c3), and the ARNT-deficient cell line c4 (B13NBii1) were obtained from the American Type Culture Collection (Manassas, VA). TCDD was from Wellington Laboratories (Guelph, ON, Canada); BNF and PCB126 from Accustandard (New Haven, CT); Power SYBR Green PCR Master Mix, TaqMan Universal PCR Master Mix, and 96-well real-time PCR plates were from Applied Biosystems (Foster City, CA). Reagents for cell culture were provided by Invitrogen (Carlsbad, CA). All other reagents were of the highest quality available from commercial sources.
In vivo experimental design
Adult (8-week old) male C57BL/6J mice from Charles River Laboratories (Montreal, QC, Canada) were housed under a 12-h light/12-h dark cycle and given food and water ad libitum. Mice were injected intraperitoneally with 3MC (80 mg/kg body weight) or corn oil vehicle. Livers were harvested 6 h after treatment and were snap-frozen in liquid nitrogen, then stored at −80 °C for later use in chromatin immunoprecipitation (ChIP) assays, RNA isolation and real-time PCR analysis, proteomic analysis and measurements of catalytic activity.
Cell culture experimental design
All hepatoma cell lines were cultured in nucleoside-free α-minimal essential medium containing 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT) and 1% penicillin/streptomycin (PEST). Cells were maintained at 37°C in a 5% CO2/air incubator with 90% humidity. Medium was changed every second day and cells were sub-cultivated once a week. For FMO induction experiments cells were seeded in 6-well plates (200,000 cells/well) and cultured for 24 h prior to treatment. Cells were exposed to the known AHR agonists: TCDD (10 nM), 3MC (1 μM), BaP (1 μM), BNF (1 μM), or PCB 126 (1 μM) for 6, 12, or 24 h. We also exposed cells to several compounds known to activate Nrf2 or oxidative stress pathways: BHA (50 μM), SUL (20 μM), menadione (10 μM), and t-BHQ (30 μM). Potential superinduction of FMO3 and CYP1A1 was studied by exposing Hepa-1 cells to10 μg/ml cycloheximide (CHX) for 1 h prior to exposure of the cells to 1 μM 3MC for 5 h or 23 h.
Real-time reverse transcription quantitative polymerase chain reaction (qPCR)
Probes and primers for mouse Fmo1
(NM_) and Fmo5
(NM_010232) were designed as reported in Celius et al., (Celius et al., 2008
). Total RNA from Hepa-1 cells was isolated using RNeasy mini kits (Qiagen, Mississauga, ON) according to the manufacturer's instructions. Real-time RT-PCR was performed using 1 μg of total RNA and a random hexamer primer in a standard procedure. Two μl of RT-reaction was used for each real-time qPCR reaction. Each reaction (25 μl) contained optimized probe and primer concentrations as well as the TaqMan universal mastermix. Expression levels of target mRNAs were normalized to the levels of 18S rRNA and analyzed using the comparative CT
(ΔΔ CT) method.
Chromatin Immunoprecipitation (ChIP) assay
ChIP assays were performed according to Matthews et al. (Matthews et al., 2005
) except that 100–120 mg of snap-frozen liver was homogenized and cross-linked in 1% formaldehyde and the DNA was isolated using a PCR purification kit (BioBasic) and eluted in 50 μl of elution buffer provided in the kit. When Hepa-1 cells were used for preparation of chromatin for ChIP assays, cells (1.8×106
) were plated in 10-cm dishes and cultured for 24 h prior to exposure to 1 μM 3MC or 1 μM BaP for 1, 6, 15 or 24 h. Cell pellets were cross-linked in 1% formaldehyde and the in vitro
ChIP assay followed the same protocol as for in vivo
ChIP assays. ChIP DNA (1 μl) was amplified by PCR with primers 5'AAGCCAAGCCAGAAAATCAA3' and 5' TCGAGGAACAGAGTGCAATG3' for the Fmo3
AH response element (AHRE); 5'-GAGGATGGAGCAGGCTTACG-3' and 5'-GGGCTACAAAGGGTGATGCTT-3' for the mouse Cyp1a1
AHRE; 5`-ATGGAAGGGTCTTGACACCA3` and 5`-CAAATTCTGCCCCATCTGTT3` for the mouse Fmo3
p53RE; and 5′-CCCGAAACCCAGGATTTTAT-3′ and 5′-GGTCTGTCCCTGACCAACT-3′ for the p53 response element in the Cdnk1a
gene (also known as p21). For real-time PCR, Power SYBR green PCR master mix was used to amplify the DNA fragments.
Protease inhibitor exposure and cell pellet preparation
Hepa-1 cells were plated (1.8×106) in a 10-cm dish and pre-exposed to protease inhibitors (5 μM MG132; 25 μM MDL28170 or 50 μM chloroquine) for 1 h followed by 23 h exposure to DMSO or 3MC. Cells were scraped and washed once with PBS and pellets were resuspended in buffer (10 mM phosphate buffer, pH 7.4, 20% glycerol, 1.0 mM EDTA, 0.2 mM PMSF) and frozen at −80°C. Cells were subsequently lysed after adding 1 μl/ml CalBiochem protease inhibitor cocktail III, by alternately freezing the cell suspension in liquid nitrogen and thawing on ice, three times. The homogenate was further degraded by passage through a 28-gauge needle 5 to 10 times. Protein concentration was measured using Pierce's Coomassie Plus assay.
Assays for FMO catalytic activity
Mouse liver microsomes (100 μg for ethionamide (ETA); 200 μg for SS) were incubated for 15 min at 37° C with 200 μM substrate and 1 mM NADPH in 0.1 M Tricine buffer at pH 8.5. Reactions were stopped by the addition of 75 μl CH3
CN (ETA) or 2 ml ethyl acetate (SS) and chilling on ice. For ETA, samples were transferred to microcentrifuge tubes and centrifuged for 30 min at 15,000 × g to remove protein and the supernatant analyzed by reverse phase HPLC (Henderson et al., 2008
) for presence of ETA S-oxide (ETASO). We assessed activity toward SS as described previously (Koukouritaki et al., 2007
): samples were vortexed for 1 min and the ethyl acetate layer removed to a new tube. The extraction was repeated and the samples taken to dryness under vacuum in a Speed Vac. After resuspension in 50:50 CH3
O, samples were analyzed by reverse phase HPLC for presence of sulindac sulfoxide (SOX). Statistical analysis was performed with GraphPad Prism 5 software unpaired two-tailed t-test. * = significant at p
AQUA mass spectrometry for mouse FMO3 protein quantification
AQUA (absolute quantification) mass spectrometry quantifies proteins by tandem mass spectrometry and MRM (multiple reaction monitoring) (Gerber et al., 2003
). AQUA employs synthetic peptide standards identical to unique tryptic peptides except that standards incorporate stable isotopes. A manuscript providing a detailed description of AQUA for quantification of mouse FMOs 1–3 and 5 will be forthcoming (MCH, SKK and DEW; in preparation); a brief description is included here.
An FMO3 standard candidate DSFPGL*NR (mouse Fmo3 amino acids 158–165) containing dual 13
C- and 15
N-labeled leucine (L*) was synthesized (Sigma, St. Louis, MO) after QTOF experiments confirmed that trypsinized microsomes from both Sf9 cells expressing mouse FMO3 and mouse liver contained the corresponding unlabeled peptide. Protein samples (25 μg/well) were separated on NuPAGE 3–8% Tris-Acetate gels (Invitrogen). Subsequently, FMO bands were excised, spiked with labeled peptide, and in-gel trypsinization performed (Shevchenko et al., 2006
) with sequencing-grade trypsin (Promega, Madison, WI).
Peptides were separated by UPLC on a Waters Symmetry C18 (180 μm × 20 mm) trapping column, and eluted onto a Waters nano Acquity UPLC column (1.7 μm, BEH130 C18, 100 μm × 100 mm) connected to a nano Acquity UPLC (Waters Corporation, Milford, MA.). Solvents were 0.1% formic acid (A) and ACN/0.1% formic acid (B), (Burdick & Jackson) at a flow rate of 0.47 μl/min with a gradient from 5–35% B. Mass spectrometry was performed using an Applied Biosystems 4000 Qtrap (Applied Biosystems/MDS Analytical Technologies, Concord, ON, Canada) using the nanospray ion source. Data was acquired in MRM mode with a 50 mSec dwell time for each transition. Precursor and product ions were 453.2 (precursor), 703.4, and 556.3 for the native protein and 456.7 (precursor), 710.4 and 563.3 for the standard.
Values obtained for area under the curve of the first product ions of sample and standard were used to calculate protein content (Analyst Software, Applied Biosystems). The second product ion provided additional confirmation of identity. The standard curve range was 50 to 1000 fmol.