Gassmann et al. on hNPCs obtained from a single male at gestational week 16. Similar results were obtained based on hNPCs from a second male at gestational week 18. For mouse neurosphere cultures, brains of wild­typ e and AhR­knockout (KO) C57/BL6 mice (Charles River Laboratories International, Wilmington, MA, USA) were removed at embryonic day (E) 15.5 (E15.5) to E17.5 and transferred to phosphate­ buffered saline. Age of the embryos was determined accord­ ing to the staging criteria of Theiler, in which E16 correspond to Theiler stage 24 (Bard et al. 1998). Brains of embryos were dissected, transferred to Dulbecco’s modi­ fied Eagle’s medium (DMEM) and mechani­ cally dissociated. Trypsin/EDTA solution was added, and the suspension was incubated for 30 min at 37°C in a humidified atmosphere. Afterward, the tissue suspension was tritu­ rated to obtain a singlec­ell suspension , which was centrifuged at 800 rpm for 5 min. Pellets were resuspended and plated in 10­ cm petri dishes. AhR deficiency of mice (Fritsche et al. 2007) was confirmed by polymerase chain reaction (PCR). Data for wild­typ e mNPC were derived from four independent prepara­ tions, and data for AhR­deficient mNPCs were obtained from two different prepara­ tions. The animals were treated humanely and with regard for alleviation of suffering. Both hNPCs and mNPCs were cultured in proliferation medium [DMEM and Hams F12 (3:1) supplemented with B27 (Invitrogen GmBH, Karlsruhe, Germany), 20 ng/mL epidermal growth factor (EGF; Biosource, Karlsruhe, Germany), 20 ng/mL recombinant human fibroblast growth factor (FGF; R&D Systems, WiesbadenN­ordenstadt, Germany), 100 U/mL penicillin, and 100 µg/mL strepto­ mycin] in a humidified 92.5% air/7.5% CO2 incubator at 37°C in suspension culture. Differentiation was initiated by growth fac­ tor withdrawal in differentiation medium [DMEM and Hams F12 (3:1) supplemented with N2 (Invitrogen), 100 U/mL penicillin, and 100 µg/mL streptomycin] and plating onto poly­ d ­ lysine (PDL)/laminin–coated chamber slides. Cell viability. We measured cell viability using a lactate dehydrogenase (LDH) assay (CytoTox­ One; Promega, Madison, WI, USA) according to the manufacturer’s instruc­ tions. Briefly, supernatants of treated cells from the migration, mRNA expression, and proliferation assessments were collected at the respective time points and incubated 2:1 with the CytoTox­One reagent for 4 hr prior to detection of fluorescence (excitation, 540 nm; emission, 590 nm). Complete lysis of cells with the included lysis buffer for 2 hr at room temperature served as a positive control. Proliferation assays, cell cycle analyses, and migration assay. Proliferation was assessed 1572 with a combination of CellTiter­Blue Assay (Promega), which measures mitochondrial reductase activity, and microscopic determina­ tion of sphere diameter as described previously (by Moors et al. 2009). We analyzed cell cycle by fluorescencea­ctivated cell sorting ( FACS). Therefore, neurospheres were exposed to the different chemicals for 48 hr, dissociated to single cells with Accutase, fixed with paraform­ aldehyde, and stained with propidium iodine (Moors et al. 2009). Spheres cultured in pro­ liferation medium with 20 ng/mL EGF and FGF served as positive controls, and spheres cultured without these growth factors served as negative controls. Migration distance was measured microscopically after 48 hr as previ­ ously described by Moors et al. (2007, 2009). Spheres exposed to 1 µM MeHgCl for 48 hr served as positive controls. Quantitative real-time PCR. NPCs were treated under differentiating conditions with 10 µM 3M­C or 1 nM TCDD, or with 0.1% dimethyl sulfoxide (DMSO) as the solvent control. After indicated time points, RNA was prepared with the Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA, USA) accord­ ing to the manufacturer’s instructions. Real­ time reverse­transcriptase PCR (RT­PCR) was performed using LightCycler instru­ mentation (Roche, Mannheim, Germany) with QuantiTect SYBR green PCR Master Mix (Qiagen, Hilden, Germany) as pre­ viously described (Fritsche et al. 2007). Conditions for PCR amplifications were as follows: initial 15 min at 95°C; 40 cycles of 15 sec at 94°C for denaturation, 25 sec of primer annealing, 30 sec at 72°C for elon­ gation; and 2 sec at 72°C for fluorescence detection. Intron­ spanning primers were designed using PRIMER 3 Software (Rozen and Skaletsky 2000) to reduce genomic amplification. Primer sequences for human ACTB (hACTB), hAhR, hAhR repressor (hAhRR), human cytochrome P450 1A1 (hCYP1A1), and hCYP1B1 were described by Fritsche et al. (2005). Other primer sequences are as follows: hARNT, forward (F), CCCTAGTCTCACCAATCGTGGAT; r e v e r s e ( R ) , G T A G C T G T T G C T C T G A T C T C C C A G ; h C - M Y C , F , A C C A C C A G C A G C G A C T C T G A ; R , T C C A G C A G A A G G T G A T C C A G A C T (56°C annealing); mouse Actb (msActb), F , C T A C A A T G A G C T G C G T G T G G ; R , T A G C T C T T C T C C A G G G A G G A ( 6 0 ° C a n n e a l i n g ) ; m s A h R , F , G A C A G T T T T C C G G C T T C T T G ; R , C G C T T C T G T A A A T G C T C T C G T ( 6 0 ° C a n n e a l i n g ) ; m s A r n t , F , T G C C T C A T C T G G T A C T G C T G ; R , G A A C A T G C T G C T C A C T G G A A ( 5 8 ° C a n n e a l i n g ) ; m s A h R R , F , G T T G G A T C C T G T A G G G A G C A ; R , A G A C C A G A G G C T C A C G C T T A ( 6 0 ° C a n n e a l i n g ) ; m s C y p 1 a 1 , F , G G C C A C T T T G A C C C T T A C A A ; R , C A G G T A A C G G A G G A C A G G A A ( 5 4 ° C a n n e a l i n g ) ; m s C y p 1 b 1 , F , A C A T G A G T T T C A G T T A T G G C C ; R , T T C C A T T C A C T G C T G A G A G C ( 5 8 ° C a n n e a l i n g ) ; m s c - m y c , T G T C C A T T C A A G C A G A C G ; F , R , GCATTTTAATTCCAGCGCATAG (54°C annealing). Expression levels were normal­ ized to the expression of β­actin . We evalu­ ated gene expression using the cycle threshold (Ct) value from each sample. Calculations are based on the ΔΔCt method (Livak and Schmittgen 2001). For determining abso­ lute copy numbers, we used product­ specific standards amplified from cDNA to generate standard curves. A target gene was consid­ ered to be quantifiable if the ratio of the copy number target gene to the copy number for βa­ctin × 1,000 exceeded 0.001. Western blotting. Proteins were isolated from hNPC or HepG2 human hepatocellular liver carcinoma cells as described by Moors et al. (2007). Whole­ cell lysates (40 µg) were separated by 10% SDSP­AGE and transferred to an Amersham Hybond­ P­membrane (GE Healthcare, Chalfont St. Giles, UK). The membrane was blocked in Trisb­uffered saline containing 0.01% Triton (vol/vol) and 5% nonfat dry milk (wt/vol) for 1 hr at room temperature, followed by incubation in the same buffer containing a polyclonal anti­ AhR antibody (1:2,000; Enzo Life Sciences, Loerrach, Germany) or an antiG­APDH anti ­ body (1:100,000; Biozol, Eching, Germany) overnight at 4°C. As secondary antibodies we used enhanced chemiluminescence (ECL) anti­ rabbit IgG or ECL antim­ouse IgG horseradish peroxidase–linked antibodies (both 1:5,000; GE Healthcare). Chemiluminescence signals were detected with Xr­ay film. Statistics. All results are presented as mean ± SEM of at least two independent experiments. We used analysis of variance combined with Dunnett’s post hoc test for multifactor analyses (concentration effects), and Student’s t­ test for two­ group compari­ sons (treatment vs. control). Significance was set at p < 0.05 (Hays 1994). Results AhR agonism or antagonism does not cause cytotoxicity in hNPCs and mNPCs. Cells were exposed to the AhR agonists 3M­C (10 µM ; a synthetic model compound for AhRa­ctivating metabolized PAH), B(a)P (10 µM; a metabo­ lized, environmentally relevant PAH), TCDD (1 nM; a nonmetabolized, environmentally relevant PAH), or the AhR antagonist MNF (1 µM; model compound). We treated hNPCs and mNPCs for 6, 12, 24, or 48 hr (differen­ tiation conditions). hNPCs were also treated for up to 14 days and mNPCs were treated for ovlume 118 | number 11 | November 2010 • Environmental Health Perspectives