Cell Culture
All cell lines and isolated cell preps were cultured in Dulbecco’s modified medium with high glucose supplemented with 10% (v/v) fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES buffer solution, 0.1 mM minimal essential medium nonessential amino acids, and penicillin-streptomycin at(0.1% DMSO). After 4 hours of treatment, cells were assayed with dual luciferaseH reporter assay system (Promega, Madison, WI). The expressed luciferase activity was measured by MicroLumat Plus luminometer (Berthold Technologies, Hartfordshire, UK). The dose-response curves and ED50 values for TCDD and SU5416 were determined using GraphPad Prism 4 software (GraphPad software Inc., La Jolla, CA). For other luciferase assays, a mouse hepatoma cell line H1L6.1c3, stably carrying a dioxin-responsive element (DRE)driven firefly luciferase reporter gene [a gift from Dr. Denison, University of California, Davis, CA [61]] was maintained with 0.3mg/ml G418 in completed DMEM media. Briefly, 0.66106 cells were seeded in each well of a six-well plate overnight and were then treated with SU5416 or other ligands at the dose described in the text. Cells were lysed by lysis buffer (Promega, Madison, WI), and the luciferase assay was performed by using a BD moonlight 3010 luminometer (BD Biosciences, San Jose, CA). The relative light unit is the indicator of luciferase expression level.resorufin product was detected using the 544 nm excitation and 590 nm emission wavelength filter set.Dose-dependent Response to SU5416 in Mice
Five-week old male C57BL/6J and DBA/2J mice were obtained from Jackson Laboratories (Bar Harbor, ME) and housed at the University of Wisconsin animal facility. Groups of four mice were orally administered 30, 80, or 120 mg of SU5416 per kg of body weight. Control groups were dosed with BNF at 120 mg/kg or an equivalent volume of corn oil (30 mL/kg). After 48 hours, liver tissue was collected for preparation of microsomes and determination of EROD activity.
Photoaffinity Ligand Binding
Hepatic cytosolic fractions were isolated from the livers of male C57BL/6J mice. Cytosolic fractions were diluted to 1 mg of protein per mL of 25 mM sodium morpholinopropane sulfonate buffer (pH 7.5) that contains 0.025% (w/v) NaN3, 1 mM EGTA, 10% (v/ v) glycerol, 15 mM NaCl, 1.0 mM dithiothreitol and 0.10% (v/v) Nonidet NP-40. A 1 nM concentration of radioligand, [125I]2azido-3-iodo-7,8-dibromodibenzo-p-dioxin (125IBr2N3DpD), was incubated with increasing concentrations of the competing compound [64]. The binding reactions were performed at 200 C for 30 min, followed by incubation at 0uC for 5 min to minimize ligand dissociation. Charcoal and gelatin (respective concentration of 1% and 0.1%, w/v) were added for 10 min at 0uC to absorb the unbound ligand and then removed by centrifugation. The 125 IBr2N3DpD ound cytosolic fractions were UV-irradiated with four Photodyne 300 nm wavelength lamps at a distance of 4 cm for 1 minute. The protein was precipitated by an overnight incubation in acetone at 220uC, collected by centrifugation and washed with cold acetone:water (9:1, v/v). The washed pellet was dissolved in sodium dodecyl sulfate sample buffer and resolved by electrophoresis on a 7.5% (w/v) polyacrylamide gel. Following staining and drying, the gel was exposed to film overnight at 280uC with an intensifying screen. The 95-kDa AHRb-1 band was excised and the amount of 125IBr2N3DpD covalently bound was quantified in a c counter.
Validation of SU5416
To assess the role of the AHR in SU5416-induced DREdependent gene transcription, the C35 AHR mutant cell line was transiently transfected with an expression vector containing the AHRb-1 cDNA (PL65), the pCH110 lacZ plasmid, and a vector containing a DRE-luciferase construct (PL265)., Control samples were mock transfected with the two reporter plasmids and the empty pSPORT vector (PL22), the parent vector from which PL65 was derived. The cells were seeded into 24-well plates at , 60% density and transfected with 67 ng of each plasmid DNA. Following 24 hours, 3 mM SU5416, 3 mM BNF or 0.3% (v/v) DMSO was added. The cells were cultured for an additional 18 hours prior to assessment of luciferase and b-galactoside activity using commercial kits (Promega, Madison, WI). The ARNTdeficient cell line, C4, was transfected, treated, and assayed as described above for the C35 cells. However, in place of the AHRbearing plasmid, these cells were transfected with the human ARNT (PL87), plus the luciferase and lacZ reporter plasmids.Isolation of Hepatic Microsomal Fraction
Hepatic microsomes were prepared by homogenizing 0.5 g of liver tissue in 5 mL of MENG buffer (25 mM buffer sodium morpholinopropane sulfonate buffer (pH 7.5) containing 0.025% (w/v) NaN3, 1 mM EGTA and 10% (v/v) glycerol). The homogenate was subjected to centrifugation at 10,0006g for 20 minutes, followed by centrifugation of the supernatant for 1 hour at 100,0006g and 4uC. The pellet was dissolved in 15 mM TrisHCl buffer containing 250 mM sucrose (pH 8.0) and aliquots were stored at 280uC. Protein concentration was determined using the Bicinchoninic Acid Protein Assay Reagent (Pierce Biotechnology, Rockford, IL).
Assessment of Ductus Venosus Status
Timed mating of female AHRfxneo/+ mice to male AHRfxneo/fxneo mice was performed [5]. At gestation day E18.5, the pregnant dams were injected i.p. with 110 mg/kg of SU5416 or the vehicle, corn oil. When the pups were 4 weeks of age, the status of the DV was determined by hepatic perfusion with Trypan Blue as previously described [5]. Briefly, each mouse was anesthetized and its liver was flushed with PBS through the cannulated portal vein. The inferior vena cava was incised to allow outflow. Trypan blue was injected through the portal vein until the liver visibly turned blue (in the case of a closed DV) or until the dye was seen exiting the IVC without perfusing the liver (when the DV is open).
High Throughput Ethoxyresorufin O-Deethylase (EROD) Analysis
High-throughput analysis of EROD activity was assayed by adapting a protocol that was described elsewhere [62,63]. Briefly, cells were seeded into 96-well plates at , 60% density and treated with the test compounds for 36 hours. The cells were washed with PBS and lysed with 30 mL water and a cycle of freeze-thaw. To each well, 150 mL of 50 mM Hepes buffer containing 26.7 mM dicumarol and 13.3 mM ethoxyresorufin were added. The samples were incubated at 30uC for 20 minutes, and 50 mL of 0.5 mM bNADPH were added to initiate the reaction. Fluorescence of theReal-time Quantitative PCR (qPCR)
Spleen was harvested from mice at the time of euthanasia and single cell suspensions were made.