mice had decreased migration capacities in culture and inhibition of signaling pathways that regulate cell migration, including focal adhesion kinase and mitogen­ activated protein kinase ERK1 (extracellular­signal regulated kinase1­) (MuleroN­avarro et al. 2005). That ERK­dependent pathways are also necessary for normal migration of hNPCs was recently shown by our group (Moors et al. 2007). However, AhR does not seem to determine ERKd­ependent migration in hNPCs, because migration distance did not change in the presence of AhR modulators (Figure 2A,C) but was impaired by ERK inhibition (Moors et al. 2007). Taken together, existing data on AhR­dependent migration imply that , as for proliferation, modulation of cell migration dif­ fers among cell types and species. To address the underlying reason for the observed species­specific differences in response to AhR modulation between hNPCs 3 2 l o r t n o c s e m iT1 0 9 8 7 l 6 o r tn5 o c s e4 m i T3 2 1 0 6 hr 12 hr 24 hr 48 hr Human AhRR Gene Mouse * AhR Arnt CYP1B1 C-Myc Specifically, in this study we discovered that proliferation of hNPCs was not affected by AhR agonists (3M­C, TCDD) or the AhR antagonist MNF (Figure 1A–C), whereas pro­ liferation of wildt­yp e mNPCs was completely blocked by AhR antagonism (Figure 1D–F). Results of the proliferation analysis in hNPCs are in contrast to previous findings obtained in human liver or neuroblastoma tumor cells that showed exogenous AhR activation by TCDD or other AhR ligands to inhibit cell proliferation and induce cell cycle arrest (Jin et al. 2004; Marlowe and Puga 2005). In con­ trast, high AhR content has been reported to promote proliferation in a human MCF breast cancer cell line that was blocked by the AhR antagonist MNF or selective AhR knockdown via small interfering RNA (Wong et al. 2009). In human umbilical vascular endothelial cells, AhR activation by 3­MC also exerted antiproliferative effects, as in the tumor cell lines (Pang et al. 2008). These data demonstrate that effects of AhR activation on cell cycle progression versus cell cycle arrest vary among different cell types, as well as between tumor and nontumor cells. In contrast to hNPCs, proliferation of mNPCs was completely blocked by MNF (Figure 1D–F). Reiners et al. (1999) reported that cell proliferation was also inhibited in the murine hepatoma cell line 1c1c7 by fla­ vone and αn­aphthoflavone treatment and in AhR­ KO cells. Moreover, mouse embryonic fibroblasts from AhR­KO mice grew more slowly than did wildt­ype cells (Elizondo et al. 2000). In contrast, AhR­KO animals show accelerated proliferation in different organs, such as skin, hair follicles, and liver blood vessels. Thus, as in humans, AhR effects on proliferation in rodent cells are also cell type dependent, and as our data indicate, cell type specificity is not consistent among different species. Besides progenitor cell proliferation, migration is another essential process in brain development. To address the role of the AhR in this process, we employed the neurosphere migration assay (Moors et al. 2007). AhR modulation by 3M­C, B(a)P, TCDD , or MNF did not affect hNPC migration (Figure 2A,C). These results were in contrast to the findings of two previous studies of human tumor cells that showed increased motility and migra­ tion of MCF7­ cells after TCDD and 3M­C treatment based on a scratch assay (Diry et al. 2006) and a transwell migration assay (Seifert et al. 2009). In contrast, we showed that AhR stimulation reduced migration of mNPCs (Figure 2B,D). This is supported by an earlier in vivo study in which prenatal exposure to the AhR ligand 7,12­ dimethylbenz[a]anthracene disrupted cerebellar cytoarchitecture in rats (Kellen et al. 1976). Conversely, immortalized mouse mammary fibroblasts from AhR­null Species-specific AhR-mediated developmental toxicity and mNPCs, we quantified copy numbers of genes that belong to the AhR machinery as well as genes that are AhR regulated. Both hNPCs and mNPCs express AhR, ARNT, AhRR, CYP1B1, and c-Myc (Figure 3A,C). However, AhR and ARNT copy numbers were close to detection limit in hNPCs (and 8–100 times lower than in mNPCs), and hAhR protein was not detectable by Western blot; consequently, only mNPCs expressed quan­ tifiable amounts of Cyp1a1 and responded to 3M­C treatment with a timer­elated induction of Cyp1a1 and Cyp1b1 mRNA (Figure 3). Moreover, low human AhR ligand affin­ ity, which is approximately 10 times lower than C57/BL6 mouse AhR­ligand attraction (Harper et al. 1988), has been attributed to an amino acid substitution in the human AhR ligand­binding domain (Ema et al. 1994). This mutation is also responsible for divergent toxic potencies of TCDD between responsive hNPC HepG2 AhR GAPDH * n25 o i ss20 e r xp15 e ld10 o f 1 2 A 1P 1 C Y 0 WT AhR-KO 3-MC TCDD AhR Arnt AhRR CYP1A1 CYP1B1 C-Myc Gene Figure 3. Induction of mRNA expression of the indicated genes in hNPCs (A) and mNPCs (C) analyzed 6, 12, 24, or 48 hr after treatment with 10 μM 3-MC under differentiating conditions. The inset in C shows Cyp1a1 expression 6 hr after 1-nM TCDD or 10-μM 3-MC exposure in wild-type (WT) and AhR-KO mNPCs. Copy numbers of the respective genes were normalized to β-actin after and are expressed as relative induction compared with vehicle control (0.1% DMSO); data are mean ± SEM of two (AhR-KO) to four independent experiments; note the different y-axis scales in A and C. (B) hNPC lysates (40 μg protein/lane) analyzed for AhR expression by Western blot using an AhR-specific antibody and HepG2 cell lysates (10 μg protein/lane) as the positive control. A GAPDH-specific antibody was used as a loading control; a representative example is shown. *p < 0.05 compared with vehicle control. Environmental Health Perspectives • volume 118 | number 11 | November 2010 1575