Chemicals and reagents.
Iscove's modified Dulbecco's medium, Moloney Murine Leukemia Virus Reverse Transcriptase, oligo(dT)18 primer, dithiothreitol (DTT), and first strand buffer were obtained from Invitrogen (Carlsbad, CA). D-Taq polymerase, 10× D-Taq buffer, and 2.5 mM dNTP were purchased from Sun Gen (Daejeon, Korea). Primary antibodies for CYP1A1, 2U1, and 2J2 and the chemiluminescence kit were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The primary antibody for actin was obtained from Cell Signaling Technology (Danvers, MA). Skimmed milk powder was obtained from BD Biosciences (San Jose, CA). Resorufin, 7-ethoxyresorufin (7-ER), SKF-525, protease inhibitor cocktail, and 3-methylenecholantheren (3-MC) were obtained from Sigma Aldrich (St. Louis, MO). Both 20-HETE and 20-HETE-d6 were obtained from Cayman (Ann Arbor, MI). All other chemicals and organic solvents for activity assays were of the highest grade available from commercial sources.
The human megakaryocytic Dami cell line was obtained from the American Type Culture Collections (Rockville, MD). Dami cells were cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cultures were maintained in a humidified atmosphere of 5% CO2 at 37°C as previously described (Khetawat et al. 2000). Dami cells were treated with 10 μM of 3-MC for 72 h in order to test the induction of CYP1A1 and determine the effect of CYP1A1 induction on ARA metabolism. For these experiments, 3-MC was dissolved in dimethyl sulfoxide (DMSO), and the final DMSO concentration in the culture medium was 0.1% (v/v). The control culture received the same volume of DMSO without 3-MC. After 3-MC treatment, the cells were incubated with 100 μM ARA for 12 h (Rehfeldt et al. 1993).
Reverse transcriptase-polymerase chain reaction.
RNAs from Dami cells, and human liver were extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Liver tissue samples were obtained from the Inje Pharmacogenomics Research Center Biobank (Inje University College of Medicine, Busan, Korea). The research protocol for the usage of human liver tissues was approved by the Institutional Review Board of Busan Paik Hospital (Inje University, Busan, Korea). Three micrograms of total isolated RNA was added to a reaction mixture containing 100 pmol oligo(dT), 2.5 mM dNTP, 0.1M DTT, 5× first strand buffer, 200 U of M-MLV reverse transcriptase and RNAase-free water for synthesis of cDNA. The reaction mixtures were incubated at 42°C for 50 min. Next, conventional PCR was performed by adding 330 ng of cDNA to a mixture containing 10 mM dNTPs, 25 mM MgCl2, 2× D-Taq polymerase buffer, 10 pmoles from each of the forward and reverse primers (Table 1), and 1.5 U of D-Taq DNA polymerase. The PCR products were separated on a 2% agarose gel and visualized using ethidium bromide staining.
Dami cell cultures were centrifuged at 1,000 rpm for 3 min. Then, lysis buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.25% Na-deoxycholate, and protease inhibitor cocktail] was added to the Dami cell pellet. The mixture was vortexed vigorously, sonicated twice for 10 s, and the resulting lysate was incubated on ice for 50 min. The lysates were stored at −80°C until use. Then, 35 μg of protein was boiled at 95°C for 5 min with 4× loading buffer containing 0.1 M Tris–HCl (pH 6.8), 4% sodium dodecyl sulfate (SDS), 1.5% bromophenol blue, 20% glycerol, and 5% β-mercaptoethanol. The denatured proteins were separated with 12% SDS–polyacrylamide gel and then transferred to a nitrocellulose membrane in a buffer containing 25 mM Tris–HCl, 192 mM glycine, and 20% (v/v) methanol. The membrane was blocked by 5% skimmed milk in Tris-Buffered Saline supplemented with 0.1% Tween 20 solution. The membrane was probed with polyclonal goat anti-CYP1A1 IgG and anti-CYP2U1 IgG, and monoclonal mouse anti-CYP2J2 IgG, separately, and reproved with monoclonal rabbit anti-beta-actin IgG at 4°C overnight. Immunoreactive proteins were detected using the enhanced chemiluminescence method according to the manufacturer's instruction (GE Healthcare Bio-Sciences, Buckinghamshire, UK).
Determination of ethoxyresorufin O-deethylase activity.
Ethoxyresorufin O-deethylase (EROD) assays were performed on Dami cells. Intact cells were incubated with 2 μg of 7-ER in a TN assay buffer [0.1 M NaCl, 50 mM Tris (pH 7.8)] for 20 min. The amount of resorufin formed was measured at excitation/emission wavelengths of 545/575 nm (Perkin Elmer Victor 3V, MTX Lab Systems, Vienna, VA). The standard curve of known concentrations of resorufin was between 50 and 500 pmoles (Kennedy et al. 1993). After measurement, the cells were lysed, and the protein concentration was determined using the Bradford dye method (Martin et al. 1995). The resurfin formation was expressed as nanomoles per minute per milligram of protein.
ARA metabolism assay.
ARA metabolism in Dami cells was investigated by incubation of 1 × 106 Dami cells with 100 μM ARA for 12 h (Rehfeldt et al. 1993). Cells were then separated by centrifugation at 1,000 rpm for 3 min. ARA metabolites were extracted from Dami cells and cell media by ethyl acetate as previously reported (Zordoky et al. 2011). ARA and its metabolites were identified and quantified using a liquid chromatography–mass spectrometry (LC-MS/MS) system, with some modifications on the previous method (Shinde et al. 2012). Briefly, metabolites were separated on a reverse-phase column Atlantis dC18 (2.1 mm i.d. × 150 mm, 3 μm particle size; Waters, Ireland) with a solvent consisting of water (A) and acetonitrile containing 0.1% formic acid (B). The flow rate of 0.25 ml/min with gradient system was as follow: 0–5 min, 5% B; 5 min, 35% B; 15 min, 65% B; 20 min, 75% B; 24–28 min, 100% B; and 28.01 min, 5% B. ARA metabolites were identified using an API 5500 mass spectrophotometer (Applied Biosystems, Foster City, CA). Peak areas for all compounds were integrated using Analyst software (version 1.2; Applied Biosystems), and 20-HETE-6 (100 μg/ml) was used as internal standard. The detection limits of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 20-HETE were 20, 10, and 20 pg/ml, respectively.
All values represent the mean ± standard deviation of triplicate reactions. Statistical significance was analyzed using a two-tailed Student’s t test. All statistical analyses were performed using the SAS program (version 9.1.3; SAS Institute, Cary, NC). Statistically significant differences as compared with the control groups are represented as *P < 0.05, **P < 0.01, and ***P < 0.001.