New insights to the role of aryl hydrocarbon receptor in bone phenotype and in dioxin-induced modulation of bone microarchitecture and material properties

https://doi.org/10.1016/j.taap.2013.09.002Get rights and content

Highlights

  • TCDD disrupts bone remodeling resulting in altered cortical and trabecular bone.

  • In trabecular bone an anabolic effect is observed.

  • Cortical bone is thinner, more porous, harder, stiffer and mechanically weaker.

  • AHR ablation results in increased trabecular bone and softer cortical bone.

  • TCDD does not affect the bones of Ahr–/– mice.

Abstract

Bone is a target for high affinity aryl hydrocarbon receptor (AHR) ligands, such as dioxins. Although bone morphology, mineral density and strength are sensitive endpoints of dioxin toxicity, less is known about effects on bone microarchitecture and material properties. This study characterizes TCDD-induced modulations of bone tissue, and the role of AHR in dioxin-induced bone toxicity and for normal bone phenotype.

Six AHR-knockout (Ahr−/−) and wild-type (Ahr+/+) mice of both genders were exposed to TCDD weekly for 10 weeks, at a total dose of 200 μg/kg bw. Bones were examined with micro-computed tomography, nanoindentation and biomechanical testing. Serum levels of bone remodeling markers were analyzed, and the expression of genes related to osteogenic differentiation was profiled using PCR array.

In Ahr+/+ mice, TCDD-exposure resulted in harder bone matrix, thinner and more porous cortical bone, and a more compact trabecular bone compartment. Bone remodeling markers and altered expression of a number of osteogenesis related genes indicated imbalanced bone remodeling. Untreated Ahr−/− mice displayed a slightly modified bone phenotype as compared with untreated Ahr+/+ mice, while TCDD exposure caused only a few changes in bones of Ahr−/− mice. Part of the effects of both TCDD-exposure and AHR-deficiency were gender dependent.

In conclusion, exposure of adult mice to TCDD resulted in harder bone matrix, thinner cortical bone, mechanically weaker bones and most notably, increased trabecular bone volume fraction in Ahr+/+ mice. AHR is involved in bone development of a normal bone phenotype, and is crucial for manifestation of TCDD-induced bone alterations.

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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