New insights to the role of aryl hydrocarbon receptor in bone phenotype and in dioxin-induced modulation of bone microarchitecture and material properties
Graphical abstract
Introduction
The quality of bone tissue is maintained through renewal of the bone by remodeling processes, in which old bone is resorbed by osteoclasts and new matrix is formed by osteoblasts (Hadjidakis and Androulakis, 2006). A number of studies have shown that bone cells are targets for exposure to high affinity aryl hydrocarbon receptor (AHR)-ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (Carpi et al., 2009, Gierthy et al., 1994, Korkalainen et al., 2009, Naruse et al., 2002, Naruse et al., 2004, Ryan et al., 2007, Singh et al., 2000). Also in vivo studies have demonstrated altered bone geometry, mineralization, material properties and strength following exposure to TCDD (Finnilä et al., 2010, Herlin et al., 2010, Hermsen et al., 2008, Jämsä et al., 2001, Miettinen et al., 2005, Nishimura et al., 2009), as well as to other dioxin-like compounds (Alvarez-Lloret et al., 2009, Andrews, 1989, Lind et al., 1999, Lind et al., 2000, Lind et al., 2004). In addition, observed skeletal alterations in wild-life have been associated with exposure to dioxin-like contaminants (Bergman et al., 1992, Lind et al., 2003, Murtomaa et al., 2007, Rodriguez-Navarro et al., 2006, Roos et al., 2010, Sonne et al., 2004).
The AHR, a ligand-activated transcriptional regulator, is ubiquitously expressed in most organs and can be activated by a structurally diverse range of chemicals (Denison and Heath-Pagliuso, 1998, Denison and Nagy, 2003, Denison et al., 2011), of which the best characterized ones include a variety of environmental contaminants. The impact of AHR as a determinant of sensitivity to TCDD-induced bone modulations has been demonstrated through the use of rat strains which differ in their AHR structure and in the sensitivity to many TCDD-induced effects (Pohjanvirta et al., 1999, Sand et al., 2010, Tuomisto et al., 1999, Viluksela et al., 2000) including alterations of bone tissue (Herlin et al., 2010, Jämsä et al., 2001, Miettinen et al., 2005). Further, AHR antagonists have been shown to prevent TCDD-induced inhibition of osteoblast differentiation in vitro (Ryan et al., 2007, Singh et al., 2000), and osteoblasts derived from AHR-knockout (Ahr−/−) mice did not show the effects of TCDD exposure observed in osteoblastic cells of wild-type (Ahr+/+) origin (Korkalainen et al., 2009). In addition to mediating TCDD-induced toxicity, AHR has a role also in normal cell and tissue homeostasis (Gonzalez and Fernandez-Salguero, 1998). Also differentiation of bone marrow derived stem cells into mature osteoblasts is reduced in AHR-deficient cells compared to cells with a normal AHR (Korkalainen et al., 2009, Ryan et al., 2007), indicating that the AHR signaling pathway plays a role also in normal bone development.
Despite the wealth of information on dioxin-induced toxicity, neither understanding of the underlying mechanism nor detailed characterization of the modulation of bone tissue is comprehensive. Bone strength is determined by its overall structure (quantity) and material integrity (quality), both of them reflecting the activity of bone cells (Donnelly, 2011). Although bone geometry, mineral density and biomechanical strength have been demonstrated as sensitive endpoints of AHR-ligand toxicity, little is known about the effects on bone microarchitecture and material properties. The present study characterizes dioxin-induced modulation of bone tissue in Ahr+/+ and Ahr−/− mice in terms of bone morphology and biomechanical properties at multiple hierarchical levels (Rho et al., 1998), as well as bone remodeling markers and osteogenesis-related gene expression. Further, the role of AHR, both in normal bone tissue properties and in dioxin-induced bone toxicity, is addressed.
Section snippets
Animals
Ahr−/− and Ahr+/+ mice in a C57BL/6J background (Schmidt et al., 1996) were obtained from The Jackson Laboratory (Bar Harbor, ME; USA) and maintained using heterozygous breeding. They were kept in a conventional laboratory animal unit subjected regularly to health surveys consisting of serological and bacteriological screening as suggested by FELASA (1996). The mice were acclimated to the experimental conditions for one week before commencing with dosing. At the start of the treatment the mice
Body weight development and general observations
TCDD exposure did not significantly affect the body weight development or body weight at the end of the treatment period in Ahr+/+ mice of either gender (data not shown). Ahr−/− mice did not differ significantly from Ahr+/+ mice, and TCDD exposure did not affect their body weight development (data not shown).
As expected (Schmidt et al., 1996), absolute and body weight normalized liver weights of Ahr−/− mice were about 25% lower than those of Ahr+/+ mice (data not shown). TCDD exposure did not
Discussion
The present study shows that exposure to TCDD modulates bone tissue at multiple hierarchical levels. In Ahr+/+ mice, TCDD exposure resulted in altered serum levels of the bone formation marker PINP and the bone resorption marker CTX, seen as a decreased PINP/CTX ratio, reflecting an imbalance in bone remodeling. In line with this finding, TCDD-exposure resulted in decreased cortical thickness and increased cortical bone porosity. In general, the morphological parameters analyzed from tibia by
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
We thank Janne Korkalainen, Ulla Naukkarinen, Arja Moilanen and Christina Trossvik for excellent technical assistance. This study was financially supported by the European Commission, Contract No QLK4-CT-2002-02528 (BONETOX), funds at Karolinska Institutet. Mikko Finnilä was supported by the Yrjö Jahnsson Foundation and the National Doctoral Programme of Musculoskeletal Disorders and Biomaterials (TBDP).
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