G2/M­phase to 2.0% ± 0.4% , whereas the distribution of cells in G0/G1­ and S­phase remained relatively constant at 51.8% ± 0.2% and 46.3% ± 0.4%, respectively. Neural cell migration is affected by AhR stimulation in mNPCs but not hNPCs. Next, we investigated whether 3M­C, B(a)P, TCDD, or MNF influences hNPC and mNPC migration. We exposed differentiating neurospheres to 1 and 10 µM 3M­C, 10 µM B(a)P, 1 nM TCDD, 0.1 µM MNF, or 1 µM MNF for 48 hr; 1 µM MeHgCl served as a positive control for inhibition of migration (Moors et al. 2009). Independent of exposures, all human neurospheres adhered to the PDL/ Human 7 days 14 days laminin matrix and migrated radially out of the sphere with an average migration distance of 739 µm ± 61 µm after 48 hr (Figure 2A,C). This was verified using hNPCs from a second individual (data not shown). In contrast to the results in hNPCs, AhR activation reduced migration distance in wild­ type mNPCs by 16% ± 5% (1 µM 3­MC), 21% ± 13% (10 µM 3M­C) , and 32% ± 10% [10 µM B(a)P] compared with solvent con­ trols, whereas migration was comparable with controls after exposure to the AhR antagonist MNF (Figure 2B,D). This effect can be attrib­ uted to AhR activation because migration of AhRd­eficient neurospheres was not disturbed Mouse WT AhR-KO * * Species-specific AhR-mediated developmental toxicity up to 7 days under proliferation conditions. Exposure times for cytotoxicity assays thereby correspond to the duration of treatments in the proliferation, migration, and gene expres­ sion assays. We chose concentrations based on known AhR activation/antagonism in other cell types. None of the exposures induced cytotoxicity (LDH release) in hNPCs or mNPCs [see Supplemental Material, Figure 1 (doi:10.1289/ehp.0901545)]. Proliferation of mNPCs, but not hNPCs, is inhibited by AhR blockage. Figure 1A and B shows quantification of the proliferation assays (sphere diameter and CellTiter­Blue Assay, respectively) of human neurospheres after exposure to 1 and 10 µM 3M­C, 1 nM TCDD, or 1 µM MNF. We found no signifi­ cant disparities in proliferation among cells with different exposures after 7 or 14 days. We verified this using hNPCs derived from a second individual (data not shown). The average diameter of the human neurospheres (excluding the negative controls cultured without growth factors) increased from 444 µm ± 15 µm to 653 µm ± 33 µm (at 7 days) and 829 µm ± 17 µm (at 14 days). The lack of exposure effects on proliferation was confirmed by FACS analyses for DNA content using propidium iodine staining (Figure 1C). We found a cell cycle distribu­ tion typical of proliferating cells in approxi­ mately 2.5% of all NPCs, as described previously (Moors et al. 2009). After 48 hr of exposure, most proliferating cells were in G0/G1p­hase (53.5% ± 3.4%), whereas 28% ± 1.8% were in S­phase, and 18.4% ± 2.8% cells were in G2/M­phase, independent of exposure conditions. We monitored proliferation of mNPCs over only 7 days because of a size­restricted halt in sphere growth beyond that time. In contrast to the hNPCs, we observed significant inhibition of mNPC proliferation after 7 days of exposure to 1 µM MNF. Although con­ trol spheres and 3M­C – and TCDDe­xposed spheres grew from an average diameter of 386 µm ± 23 µm to 551 µm ± 85 µm, MNF­ exposed spheres remained at 405 µm ± 78 µm after 7 days. This was due to AhR inhibition, because proliferation of AhRd­eficient neuro ­ spheres was not affected by MNF treatment (Figure 1D,E). Effects of MNF on mNPC proliferation were confirmed by FACS analy­ ses for DNA content using propidium iodine staining (Figure 1F). As for hNPCs, we found a cell cycle distribution typical of proliferat­ ing cells in approximately 2.5% of all cells. In the positive control (solvent plus growth factors), 56.1% ± 4.6% of these cells were in G0/G1p­hase, 38.1% ± 4.3% were in Sp­hase, and 5.8% ± 0.4% cells were in G2/M­phase after 48 hr of exposure. 3­MC –treated cells showed a similar distribution. However, MNF treatment reduced the number of cells in Figure 1. MNF inhibits proliferation of mNPCs (D–F) but not hNPCs (A–C). Abbreviations: NC, negative con- trol (0.1% DMSO without added growth factors); PC, positive control (0.1% DMSO plus 20 ng/mL each of EGF and FGF); WT, wild type. After exposure of NPCs to 3-MC (1 μM and 10 μM), TCDD (0.001 μM), or MNF (1 μM) for 7 (mNPCs) or 14 (hNPCs) days, sphere diameters and mitochondrial reductase activities of human (A, B) and WT and AhR-KO mouse (D, E) neurospheres were measured; results are reported as percentages of corresponding measurements in PCs. The results for each end point were plotted in separate x,y-diagrams, and the gradient of the linear regression curve was assessed; data represent the mean gradient ± SEM of two (AhR-KO) to four independent experiments (5–6 spheres/exposure). Cell cycle phase distributions (values are shown within or above bars) of dissociated, fixed, and propidium iodine–stained hNPCs (C) and mNPCs (F) analyzed by FACS. Data represent the mean ± SEM of three independent experiments after 48-hr exposure to 10 μM 3-MC or 1 μM MNF. *p < 0.05 compared with PCs. Environmental Health Perspectives • volume 118 | number 11 | November 2010 1573 150 )125 % (r100 e t e iam75 d re 50 e h pS25 150 )125 % ( ity100 v i t ac 75 e s tca 50 u d eR25 125 )100 % ( tn 75 e t n c o 50 A N D25 0 * * 0 PC NC 1 10 1 NC 1 10 0.001 1 3-MC MNF 3-MC TCDD MNF Treatment (µM) 20.2 29.8 50.0 PC G1 S 15.2 28.0 56.8 19.9 26.3 53.7 3-MC MNF G2/M * 4.3 9.9 86.7 NC 125 100 ) (%75 r te e 50 m ida 25 e re 0 h p S–25 –50 125 )100 (%75 y t iiv 50 t ac 25 e tsa 0 c du–25 e R–50 –75 125 )100 % ( tn 75 e t n co 50 A N D25 0 * * * * PC NC 1 10 0.001 1 3-MC TCDD MNF Treatment (µM) 5.8 38.1 56.1 PC G1 S 6.6 41.7 51.7 * 2.0 46.2 51.8 G2/M * 0.8 20.3 78.9 3-MC MNF NC 0 PC NC 1 10 1 NC 1 10 0.001 1 3-MC MNF 3-MC TCDD MNF Treatment (µM) 7 days 14 days PC NC 1 10 0.001 1 3-MC TCDD MNF Treatment (µM) WT AhR-KO