Inhibitory effects on osteoblast differentiation in vitro by the polychlorinated biphenyl mixture Aroclor 1254 are mainly associated with the dioxin-like constituents Maria Herlin a,b , Mattias Öberg a , Joakim Ringblom a , Bertrand Joseph c , Merja Korkalainen d , Matti Viluksela d,e , Rachel A. Heimeier a , Helen Håkansson a,⇑ a Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden b Clinical and Molecular Osteoporosis Research Unit, Department of Clinical Sciences Malmö, Lund University, Malmö, Sweden c Department of Oncology-Pathology, Cancer Centrum Karolinska, Karolinska Institutet, Stockholm, Sweden d Department of Environmental Health, National Institute for Health and Welfare, Kuopio, Finland e Department of Environmental Science, University of Eastern Finland, Kuopio, Finland article info Article history: Received 7 July 2014 Accepted 1 March 2015 Available online 17 March 2015 Keywords: Osteoblast TCDD Aroclor 1254 Aryl hydrocarbon receptor Toxic equivalency factors Mixture toxicity abstract The polychlorinated biphenyl (PCB) mixture Aroclor 1254 alters bone tissue properties. However, the mechanisms responsible for the observed effects have not yet been clarified. This study compared the effect of Aroclor 1254 on the expression of osteoblast differentiation markers in MC3T3-E1 cells with the corresponding effect of the dioxin reference compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and two PCB congeners belonging to the category of non-dioxin-like PCBs. The aim of the study was to quantify the relative influence of dioxin-like and non-dioxin-like PCB-components on osteoblast differ- entiation. Expression of marker genes for AhR activity and osteoblast differentiation were analyzed, and relative potency (REP) values were derived from Benchmark concentration-effect curves. Expression of alkaline phosphatase and osteocalcin were decreased by both Aroclor 1254 and TCDD expo- sure, while the PCB-congeners PCB19 and PCB52 slightly induced the expression. The relative potency of Aroclor 1254 for inhibitory effects on osteoblast differentiation marker genes was within the expected range as estimated from the chemical composition of Aroclor 1254. These results are consistent with pre- viously observed bone modulations following in vivo exposure to Aroclor 1254 and TCDD, and demon- strate that the inhibitory effects of Aroclor 1254 on osteoblast differentiation by the dioxin-like constituents are over-riding the contribution of non-dioxin-like PCBs. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction Experimental studies have demonstrated that exposure to the potent aryl hydrocarbon receptor (AhR) ligand 2,3,7,8-tetra- chlorodibenzo-p-dioxin (TCDD) affects bone geometry, densitome- try and biomechanical properties (Alvarez-Lloret et al., 2009; Finnila et al., 2010; Herlin et al., 2010, 2013; Hermsen et al., 2008; Jamsa et al., 2001; Lind et al., 1999, 2000, 2004, 2009; Miettinen et al., 2005; Nishimura et al., 2009). TCDD also inhibits the differentiation of osteoblasts (Carpi et al., 2009; Korkalainen et al., 2009; Ryan et al., 2007; Singh et al., 2000) and osteoclasts (Korkalainen et al., 2009) in vitro. The AhR is ubiquitously expressed in most organs and cells in the body, including osteo- blasts and osteoclasts (Ilvesaro et al., 2005), and data from AhR- knockout mice, which show a partly different bone phenotype compared to wild-type mice (Herlin et al., 2013), suggest that AhR has a role in normal bone development. Further, most bone tissue modulations induced by TCDD in wild-type mice were not observed in AhR-knockout mice (Herlin et al., 2013). Similarly, effects of TCDD on osteoblasts derived from wild-type mice were not seen in osteoblasts from AhR-knockout mice (Korkalainen et al., 2009). These data strongly support that AhR plays a major role in the observed effects TCDD on bone properties. Bone tissue is continuously undergoing remodeling; a regulated process that is essential for the maintenance of bone size, shape and quality (Hadjidakis and Androulakis, 2006). The balance between bone resorption and formation is dependent on complex interactions between various cell types as well as local and systemic factors. Imbalance in the regulation of bone remodeling http://dx.doi.org/10.1016/j.tiv.2015.03.006 0887-2333/Ó 2015 Elsevier Ltd. All rights reserved. ⇑ Corresponding author at: Institute of Environmental Medicine, Karolinska Institutet, P.O. Box 210, SE-171 77 Stockholm, Sweden. Tel.: +46 8 5248 7527; fax: +46 8 34 38 49. E-mail address: Helen.Hakansson@ki.se (H. Håkansson). Toxicology in Vitro 29 (2015) 876–883 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit