Gassmann et al. by 3M­C or B(a)P (Figure 2D). Interestingly, TCDD did not disrupt wild­ type mNPC migration despite AhR activation (Figure 2D). This might be due to the fact that, in con­ trast to 3­MC and B(a)P , TCDD is hardly metabolized and thus does not produce reactive intermediates. AhR-dependent gene transcription. AhR­ dependent gene transcription is inducible only in mNPCs, and not in hNPCs because of low abundance of AhR and ARNT tran­ scripts and absence of AhR protein in human cells. Because 3­MC, B(a)P , and MNF did not influence hNPC viability, proliferation, or migration but did modulate proliferation or migration of mNPCs, we determined AhR and ARNT mRNA expression after exposure to these compounds in hNPCs and mNPCs under proliferating and differentiating condi­ tions. AhR and ARNT mRNAs were expressed at very low copy numbers in hNPCs (0.6–2.5 and 13/1,000 copies β­actin, respectively) and higher copy numbers in mNPCs (20–63 and 716–1,045/1,000 copies β­actin , respec­ tively) (Table 1). In addition, CYP1A1 expres­ sion was undetectable in untreated hNPCs. That β­actin was in this case valid to use as a housekeeping gene was demonstrated by * * * Mouse WT AhR-KO Human ) ) % % o o i i t t M M * 1 125 100 (n 75 irga 50 25 0 n a m u H e s u o M 125 100 (n 75 irga 50 25 0 Gene AhR ARNT AhRR CYP1A1 CYP1B1 C-MYC 1574 ovlume 118 | number 11 | November 2010 • Environmental Health Perspectives * 1 MeHgCI Diff 102.12 77.13 638.85 ND 5.29 14990.75 normalization of proliferating versus differ­ entiating NPCs to three additional house­ keeping genes [RPL27, RPL30, OAZ1; see Supplemental Material, Figure 2 (doi:10.1289/ ehp.0901545)]. Expression of the AhR target genes AhRR, CYP1A1, CYP1B1, and c-myc was not significantly induced by 10 µM 3M­C after 6, 12, 24, and 48 hr of differentiation in hNPCs (Figure 3A). We obtained comparable results for hNPCs from a second individual after 6 hr of treatment (data not shown). In contrast, 6 hr of exposure to 10 µM 3­MC significantly induced Cyp1a1 and Cyp1b1 mRNA in wild­ type mNPCs to levels 6.6 ± 1.7 and 2.5 ± 0.25 times higher than con­ trols, respectively (Figure 3C). Although 1 nM TCDD did not disturb neural migration, it induced AhR signaling in wildt­yp e mNPCs, increasing Cyp1a1 expression 21 times relative to controls (Figure 3C, inset). Comparison of mRNA expression lev­ els between hNPCs and mNPCs showed that genes belonging to the AhR machin­ ery and AhR­ dependent genes were gener­ ally expressed in higher copy numbers/1,000 copies β­ actin in mNPCs than in hNPCs (Table 1). Lack of AhR protein in hNPCs was confirmed by Western blot (Figure 3B). These results demonstrate that AhR signaling pathway gene products mediate the effects of the AhR agonists 3­ MC and B(a)P and the AhR antagonist MNF on proliferation and migration in mNPCs, because AhR­deficient mNPCs are protected against these effects (Figure 3C, inset). Discussion The development of cellb­ased, non animal test­ ing strategies for hazard assessment of chemicals is currently one of the most important tasks in toxicological research. In this regard, it is most important to choose appropriate model systems that are truly predictive for humans (Krewski et al. 2009; National Research Council 2007). Human tumor cell lines that are easily acces­ sible in large quantities bear the restriction that they do not represent cellular metabolism and signal transduction of normal cells. In con­ trast, primary cells are often obtained as ex vivo cultures from rodents. Such primary cultures are regarded as superior to tumor­derived cells. However, species­specific differences limit their application. One example of how rodent primary cells can indeed misclassify hazards for humans is provided by this study, which shows mouse­derived primary cells to be more susceptible to AhR modulation than their human counterparts. With regard to chemical testing, it is critical to be aware of such differences in order to avoid over­ or underestimating hazards that chemicals pose to humans, and thereby protect human health and allow industry production and develop­ ment of chemicals at the same time. DMSO 1 10 10 0.001 0.1 1 DMSO 1 10 10 0.001 0.1 1 3-MC B(a)P TCDD MNF MeHgCI 3-MC B(a)P TCDD MNF Treatment (µM) Treatment (µM) Figure 2. AhR agonists shorten wild-type mNPC but not hNPC migration. hNPCs (A and C) and wild-type (B and D) and AhR-KO (D) mNPCs were exposed to 3-MC, B(a)P, TCDD, MNF, or MeHgCl (PC) during differen- tiation for 48 hr. Migration distance from the edge of the sphere to the furthest outgrowth was measured. Data represent means ± SEs of two (AhR-KO) to five independent experiments (5–8 spheres/exposure). Bar = 500 μm. *p < 0.05 compared with 0.1% DMSO. Table 1. Comparison of human and mouse mRNA copy numbers/1,000 copies of β-actin in proliferating and 24-hr differentiating NPCs.a Human Mouse Mouse:human ratio Prolif 2.45 12.95 0.94 < 0.001 0.01 5559.59 Diff 0.62 13.55 0.61 < 0.001 0.06 3017.13 Prolif 20.08 716.26 79.21 18.63 146.24 14835.55 Diff 63.21 1045.07 387.55 31.64 834.91 15967.72 Prolif 8.19 55.31 84.24 ND 16407.23 2.67 Abbreviations: Diff, differentiating; ND, not detectable; Prolif, proliferating. aData represent at least three independent experiments. The mouse:human ratio is shown to compare human and mouse mRNA expression levels.