Multipotent mesenchymal stem cells (MSCs) maintain the ability to differentiate into adipogenic, chondrogenic, or osteogenic cell lineages. There is increasing concern that exposure to environmental agents such as aryl hydrocarbon receptor (AhR) ligands, may perturb the osteogenic pathways responsible for normal bone formation. The objective of the current study was to evaluate the potential of the prototypic AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to disrupt osteogenic differentiation of human bone-derived MSCs (hBMSCs) in vitro. Primary hBMSCs from three donors were exposed to 10 nM TCDD and differentiation was interrogated using select histological, biochemical, and transcriptional markers of osteogenesis. Exposure to 10 nM TCDD resulted in an overall consistent attenuation of alkaline phosphatase (ALP) activity and matrix mineralization at terminal stages of differentiation in primary hBMSCs. At the transcriptional level, the transcriptional regulator DLX5 and additional osteogenic markers (ALP, OPN, and IBSP) displayed attenuated expression; conversely, FGF9 and FGF18 were consistently upregulated in each donor. Expression of stem cell potency markers SOX2, NANOG, and SALL4 decreased in the osteogenic controls, whereas expression in TCDD-treated cells resembled that of undifferentiated cells. Coexposure with the AhR antagonist GNF351 blocked TCDD-mediated attenuation of matrix mineralization, and either fully or partially rescued expression of genes associated with osteogenic regulation, extracellular matrix, and/or maintenance of multipotency. Thus, experimental evidence from this study suggests that AhR transactivation likely attenuates osteoblast differentiation in multipotent hBMSCs. This study also underscores the use of primary human MSCs to evaluate osteoinductive or osteotoxic potential of chemical and pharmacologic agents in vitro.

Emerging evidence demonstrates the importance of sufficient vitamin D (1α, 25-dihydroxyvitamin D3) levels during early life stage development with deficiencies associated with long-term effects into adulthood. While vitamin D has traditionally been associated with mineral ion homeostasis, accumulating evidence suggests non-calcemic roles for vitamin D including metabolic homeostasis. In this study, we examined the hypothesis that vitamin D deficiency (VDD) during early life stage development precedes metabolic disruption. Three dietary cohorts of zebrafish were placed on engineered diets including a standard laboratory control diet, a vitamin D null diet, and a vitamin D enriched diet. Zebrafish grown on a vitamin D null diet between 2-12 months post fertilization (mpf) exhibited diminished somatic growth and enhanced central adiposity associated with accumulation and enlargement of visceral and subcutaneous adipose depots indicative of both adipocyte hypertrophy and hyperplasia. VDD zebrafish exhibited elevated hepatic triglycerides, attenuated plasma free fatty acids and attenuated lipoprotein lipase activity consistent with hallmarks of dyslipidemia. VDD induced dysregulation of gene networks associated with growth hormone and insulin signaling, including induction of suppressor of cytokine signaling. These findings indicate that early developmental VDD impacts metabolic health by disrupting the balance between somatic growth and adipose accumulation.

Abstract Background The farnesoid X receptor (FXR), pregnane X receptor (PXR), and vitamin D receptor (VDR) are three closely related nuclear hormone receptors in the NR1H and 1I subfamilies that share the property of being activated by bile salts. Bile salts vary significantly in structure across vertebrate species, suggesting that receptors binding these molecules may show adaptive evolutionary changes in response. We have previously shown that FXRs from the sea lamprey (Petromyzon marinus) and zebrafish (Danio rerio) are activated by planar bile alcohols found in these two species. In this report, we characterize FXR, PXR, and VDR from the green-spotted pufferfish (Tetraodon nigriviridis), an actinopterygian fish that unlike the zebrafish has a bile salt profile similar to humans. We utilize homology modelling, docking, and pharmacophore studies to understand the structural features of the Tetraodon receptors. Results Tetraodon FXR has a ligand selectivity profile very similar to human FXR, with strong activation by the synthetic ligand GW4064 and by the primary bile acid chenodeoxycholic acid. Homology modelling and docking studies suggest a ligand-binding pocket architecture more similar to human and rat FXRs than to lamprey or zebrafish FXRs. Tetraodon PXR was activated by a variety of bile acids and steroids, although not by the larger synthetic ligands that activate human PXR such as rifampicin. Homology modelling predicts a larger ligand-binding cavity than zebrafish PXR. We also demonstrate that VDRs from the pufferfish and Japanese medaka were activated by small secondary bile acids such as lithocholic acid, whereas the African clawed frog VDR was not. Conclusions Our studies provide further evidence of the relationship between both FXR, PXR, and VDR ligand selectivity and cross-species variation in bile salt profiles. Zebrafish and green-spotted pufferfish provide a clear contrast in having markedly different primary bile salt profiles (planar bile alcohols for zebrafish and sterically bent bile acids for the pufferfish) and receptor selectivity that matches these differences in endogenous ligands. Our observations to date present an integrated picture of the co-evolution of bile salt structure and changes in the binding pockets of three nuclear hormone receptors across the species studied.

Research suggests that thyroid cancer incidence rates are increasing, and environmental exposures have been postulated to be playing a role. To explore this possibility, we conducted a pilot study to investigate the thyroid disrupting bioactivity of chemical mixtures isolated from personal silicone wristband samplers within a thyroid cancer cohort. Specifically, we evaluated TRβ antagonism of chemical mixtures extracted from wristbands (n = 72) worn by adults in central North Carolina participating in a case-control study on papillary thyroid cancer. Sections of wristbands were solvent-extracted and analyzed via mass spectrometry to quantify a suite of semivolatile chemicals. A second extract from each wristband was used in a bioassay to quantify TRβ antagonism in human embryonic kidney cells (HEK293/17) at concentrations ranging from 0.1 to 10% of the original extract (by volume). Approximately 70% of the sample extracts tested at a 1% extract concentration exhibited significant TRβ antagonism, with a mean of 30% and a range of 0-100%. Inhibited cell viability was noted in >20% of samples that were tested at 5 and 10% concentrations. Antagonism was positively associated with wristband concentrations of several phthalates, organophosphate esters, and brominated flame retardants. These results suggest that personal passive samplers may be useful in evaluating the bioactivities of mixtures that people contact on a daily basis. We also report tentative associations between thyroid receptor antagonism, chemical concentrations, and papillary thyroid cancer case status. Future research utilizing larger sample sizes, prospective data collection, and measurement of serum thyroid hormone levels (which were not possible in this study) should be utilized to more comprehensively evaluate these associations.