Research Species-Specific Differential AhR Expression Protects Human Neural Progenitor Cells against Developmental Neurotoxicity of PAHs Kathrin Gassmann,1 Josef Abel,1 Hanno Bothe,1 Thomas Haarmann-Stemmann,1 Hans F. Merk,2 Kim N. Quasthoff,1 Thomas Dino Rockel,1 Timm Schreiber,1 and Ellen Fritsche 1,2 1Department of Molecular Toxicology, Institut für umweltmedizinische Forschung gGmbH, Heinrich Heine University, Düsseldorf, Germany; 2Department of Dermatology, University Hospital, RWTH Aachen, Aachen, Germany Background : Because of their lipophilicity, persistent organic pollutants (POPs) cross the human placenta, possibly affecting central nervous system development. Most POPs are known aryl hydro- carbon receptor (AhR) ligands and activators of AhR signaling. Therefore, AhR activation has been suggested to cause developmental neurotoxicity (DNT). o Bjectiv : We studied the effects of AhR ligands on basic processes of brain development in two comparative in vitro systems to determine whether AhR-activation is the underlying mechanism for reported DNT of POPs in humans. Msdohte : We employed neurosphere cultures based on human neural progenitor cells (hNPCs) and wild-type and AhR-deficient mouse NPCs (mNPCs) and studied the effects of different AhR agonists [3-methylcholanthrene (3-MC), benzo(a)pyrene [B(a)P], and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)] and an antagonist [3´-methoxy-4´-nitroflavone (MNF)] on neurosphere development. Moreover, we analyzed expression of AhR and genes involved in AhR signaling. r esult : In contrast to wild-type mNPCs, hNPCs and AhR-deficient mNPCs were insensitive to AhR agonism or antagonism. Although AhR modulation attenuated wild-type mNPC proliferation and migration, hNPCs and AhR-deficient mNPCs remained unaffected. Results also suggest that species-specific differences resulted from nonfunctional AhR signaling in hNPCs. c onclusi : Our findings suggest that in contrast to wild-type mNPCs, hNPCs were protected against polycyclic aromatic hydrocarbon–induced DNT because of an absence of AhR. This differ- ence may contribute to species-specific differences in sensitivity to POPs. k ey words : AhR, AhR knockout, DNT, neural progenitor cells, neurospheres, PAH. Environ Health Perspect 118:1571–1577 (2010). doi:10.1289/ehp.0901545 [Online 22 June 2010] Persistent organic pollutants (POPs) bio­ accumulate through the food chain and may cause adverse effects on human health and the environment. Main substance classes are poly­ cyclic aromatic hydrocarbons (PAHs), such as 3­ methylcholanthrene (3­ MC) and benzo(a) pyrene [B(a)P], dioxins such as 2,3,7,8­tet ­ rachlorodibenzo­ p­dioxin (TCDD), and polychlorinated biphenyls (PCBs). Because of their lipophilicity, POPs cross the human placenta, exposing the fetus to the contami­ nant body burden of the mother. This may result in adverse health outcomes, includ­ ing effects on central nervous system (CNS) development (reviewed by Wormley et al. 2004). One potential source for in utero expo­ sure to POPs, especially PAHs, is maternal cigarette smoking during pregnancy (Polanska et al. 2009; Rodgman et al. 2000). An inverse relationship between maternal smoking dur­ ing pregnancy, offspring intelligence (intel­ ligence quotient), and cognitive ability has been described in numerous cohort studies (Batty et al. 2006; Fried et al. 1998; Olds et al. 1994). Additionally, behavioral problems and psychiatric disorders in offspring have been associated with smoking during pregnancy (Fergusson et al. 1998; Thapar et al. 2003; Weissman et al. 1999). In addition to smok­ ing, in utero exposure to PCBs and/or diox­ ins has been associated with cognitive deficits in children (reviewed by Grandjean and Landrigan 2006; Jacobson and Jacobson 1997; Patandin et al. 1999). It has been proposed that etiological mechanisms involve the POP­ activated aryl hydrocarbon receptor (AhR), an evolutionary highly conserved member of the basic helix­loop­helix /Per­ARNT­Sim (bHLH/PAS) family of transcription factors (reviewed by Kakeyama and Tohyama 2003). Ligand binding to this cytosolic receptor induces nuclear translocation (Gasiewicz and Bauman 1987) and heterodimerization with another bHLH/PAS protein, ARNT (AhR nuclear translocator) (Reyes et al. 1992). The resulting complex recognizes and binds spe­ cific DNA sequences (i.e., dioxin­responsive elements) within gene promoter regions and modulates subsequent transcription of AhR­ dependent genes (FujisawaS­ehara et al. 1987). Many POPs, including PAHs and dioxins, are known AhR ligands and activators of AhR signaling. It has therefore been proposed that AhR activation causes developmental neuro­ toxicity (DNT) (Williamson et al. 2005). This hypothesis is supported by studies in inverte­ brate (Caenorhabditis elegans) and vertebrate (zebrafish, chicken, rat, monkey) species in which dioxins and related compounds cause morphological abnormalities of the brain or deficits in cognition and/or behavior (Henshel et al. 1997; Hill et al. 2003; reviewed by Kakeyama and Tohyama 2003; Qin and Powell­Coffman 2004). Whether AhR Environmental Health Perspectives • volume 118 | number 11 | November 2010 activation is the underlying mechanism for the reported DNT effects after POP exposure in humans is not known. To investigate potential speciess­pecific dif ­ ferences, we employed comparative in vitro test systems for brain development based on neurosphere cultures from human and mouse neural progenitor cells (hNPCs and mNPCs, respectively). These three­dimensional cell systems mirror basic processes of fetal brain development such as proliferation, migration, differentiation, and apoptosis. Moreover, they detect developmental neurotoxicants in vitro (Fritsche et al. 2005; Moors et al. 2007, 2009). We report here that, in contrast to wildt­yp e mNPCs, hNPCs and AhR­deficient mNPCs were insensitive to AhR agonism or antagonism due to nonfunctional AhR signaling, which suggests that humans are protected from AhR­ dependent, POP­induced DNT. Knowledge about such species­specific differences is of utmost importance regarding chemical testing and hazard assessment for humans. Materials and Methods Chemicals. The AhR antagonist 3´­ methoxy­ 4´­ nitroflavone (MNF) was kindly provided by G. Vielhaber (Symrise, Holzminden, Germany). Methylmercury chloride (MeHgCl) was obtained from Riedel­ de Haën (Seelze, Germany), and TCDD was purchased from LGC Standards (Wesel, Germany). All addi­ tional chemicals used (unless otherwise noted) were purchased from SigmaA­ldrich (Munich, Germany) and were of the highest purity available. Cell culture. hNPCs used in this study were purchased from Lonza Verviers SPRL (Verviers, Belgium). Data presented in this study are based on experiments conducted Address correspondence to E. Fritsche, Institut für umweltmedizinische Forschung gGmbH, Heinrich Heine University, Department of Molecular Toxicology, Auf’m Hennekamp 50, 40225 Dusseldorf, Germany. Telephone: 00492113389217. Fax: 00492113190910. E­mail: ellen.fritsche@ unid­uesseldorf.de Supplemental Material is available online (doi:10. 1289/ehp.0901545 via http://dx.doi.org/). This work was supported by the German Research Foundation (GRK1427), the German Ministry for the Environment, the German Ministry for Education and Research, and the Cefic LRI program. The authors declare they have no actual or potential competing financial interests. Received 7 October 2009; accepted 22 June 2010. 1571