Presenting Author:

Soyeon Jeong, M.S.

Principal Investigator:

Erin Hsu, Ph.D.

Department:

Orthopaedic Surgery

Keywords:

dioxin, osteogenesis, environmental contaminant, cigarette smoke, aryl hydrocarbon receptor

Location:

Third Floor, Feinberg Pavilion, Northwestern Memorial Hospital

B126 - Basic Science

Phytochemical protection against the anti-osteogenic effects of dioxin

Inhibition of bone healing and increased rates of pseudarthrosis are well-established effects associated with cigarette smoking, but the underlying mechanisms are not well understood. Previous studies have implicated the Aryl Hydrocarbon Receptor (Ahr) as an inhibitor of osteogenic differentiation, but the mechanisms are not well-established. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a prototype Ahr ligand constituent of cigarette smoke, as well as an industrial by-product of chlorinated compound combustion, and contaminent found in foods such as meat, dairy, and fish. It has been implicated in anti-osteogenic effects. The purpose of this study was to elucidate the effects of dioxin on osteogenic differentiation of human MG-63 osteoblast-like cells and to determine whether these effects could be mitigated by co-treatment with Ahr antagonists. MG-63 human cells were cultured under standard or osteogenic conditions. The effects of dioxin and/or the Ahr antagonists -naphthoflavone (ANF) and resveratrol (found in grapes and red wine), and 3,3’-diindolylmethane (DIM; found in cruciferous vegetables) on osteogenic gene and protein expression were quantified. Factors critical for osteogenesis, such as cell adhesion, directional migration, and mineral deposition were also quantified. Dioxin induced CYP1A1 expression and ethoxyresorufin-o-deethylase (EROD) activity, demonstrative of a functional Ahr in MG-63 cells. Pre-treatment of cells grown with dioxin under osteogenic conditions caused a significant reduction of ALP activity and matrix mineralization. Cell adhesion to fibronectin-coated plastic as well as directional cell migration towards a FBS gradient were also reduced after dioxin exposure. QPCR and western blotting showed reduced expression of ALP, OCN, RUNX2, CXCL12, PHEX, and OCN, as well as integrinα5. All three antagonists increased CXCL12 whereas DIM significantly increased CXCR4 expression. All three antagonists reduced CYP1A1 expression to near baseline levels as well as significantly increased RUNX2 expression and cell migration. ANF and resveratrol both increased ALP expression, ALP activity, and mineralization. The cumulative effects of Ahr activation and bone regeneration are not currently well understood. Our results suggest that dioxin-like compounds-of which there are dozens in cigarette smoke and the environment-may have a direct effect on bone healing. The Ahr pathway is the primary mode by which dioxin exerts its toxic effects, and co-administration of Ahr antagonists diminished the impact dioxin had on MG-63 cells. Dioxin has an extremely long half-life in humans (7-10 years), that could have a long lasting impact on bone’s regenerative capacity. Futhur studies investigating the effects of Ahr antagonists may lead to development of a viable therapeutic approach to combat dioxin’s inhibitory effects. This could improve the surgical outcomes of patients who smoke, or have environmental or dietary exposure.