Endocrine Disruptor-Induced Bone Damage Due to Hormone Dysregulation: A Review.

Hormones are indispensable for bone development, growth, and maintenance. While many of the genes associated with osteogenesis are well established, it is the recent findings in endocrinology that are advancing the fields of bone biology and toxicology. Endocrine-disrupting chemicals (EDCs) are defined as chemicals that interfere with the function of the endocrine system. Here, we report recent discoveries describing key hormone pathways involved in osteogenesis and the EDCs that alter these pathways. EDCs can lead to bone morphological changes via altering hormone receptors, signaling pathways, and gene expression. The objective of this review is to highlight the recent discoveries of the harmful effects of environmental toxicants on bone formation and the pathways impacted. Understanding the mechanisms of how EDCs interfere with bone formation contributes to providing a comprehensive toxicological profile of a chemical.

Sidestream Smoke Extracts from Harm-Reduction and Conventional Camel Cigarettes Inhibit Osteogenic Differentiation via Oxidative Stress and Differential Activation of intrinsic Apoptotic Pathways.

Epidemiological studies suggest cigarette smoking as a probable environmental factor for a variety of congenital anomalies, including low bone mass, increased fracture risk and poor skeletal health. Human and animal in vitro models have confirmed hypomineralization of differentiating cell lines with sidestream smoke being more harmful to developing cells than mainstream smoke. Furthermore, first reports are emerging to suggest a differential impact of conventional versus harm-reduction tobacco products on bone tissue as it develops in the embryo or in vitro. To gather first insight into the molecular mechanism of such differences, we assessed the effect of sidestream smoke solutions from Camel (conventional) and Camel Blue (harm-reduction) cigarettes using a human embryonic stem cell osteogenic differentiation model. Sidestream smoke from the conventional Camel cigarettes concentration-dependently inhibited in vitro calcification triggered by high levels of mitochondrially generated oxidative stress, loss of mitochondrial membrane potential, and reduced ATP production. Camel sidestream smoke also induced DNA damage and caspase 9-dependent apoptosis. Camel Blue-exposed cells, in contrast, invoked only intermediate levels of reactive oxygen species insufficient to activate caspase 3/7. Despite the absence of apoptotic gene activation, damage to the mitochondrial phenotype was still noted concomitant with activation of an anti-inflammatory gene signature and inhibited mineralization. Collectively, the presented findings in differentiating pluripotent stem cells imply that embryos may exhibit low bone mineral density if exposed to environmental smoke during development.

Exposure to Cigarette Smoke Impedes Human Osteoblast Differentiation Independently of Nicotine.

INTRODUCTION: Tobacco smoking has been implicated in an array of adverse health outcomes, including those that affect adult bone. However, little is known about the impact of tobacco products on developing bone tissue as it develops in the embryo. AIMS AND METHODS: Here, human embryonic stem cells were differentiated into osteoblasts in vitro and concomitantly exposed to various concentrations of smoke solutions from two conventional, one additive-free and two harm-reduction brands of cigarettes. Differentiation inhibition was determined by calcium assays that quantified matrix mineralization and compared to the cytotoxicity of the tobacco product. RESULTS: Exposure to mainstream smoke from conventional and additive-free cigarettes caused no inhibition of cell viability or mineralization, while sidestream smoke (SS) concentration-dependently produced cell death. In contrast, mineralization was inhibited only by the highest mainstream concentration of harm-reduction smoke solution. Additionally, sidestream smoke solution from the harm-reduction cigarettes impeded calcification at concentrations lower than those determined to be cytotoxic for conventional products. CONCLUSIONS: Sidestream smoke impaired in vitro osteogenesis at subtoxic concentrations. In addition, though often perceived as safer, smoke from harm-reduction cigarettes was more potent in inhibiting in vitro osteogenesis than smoke from conventional cigarettes. IMPLICATIONS: This study adds to a growing list of adverse outcomes associated with pre-natal tobacco exposure. Specifically, in vitro exposure to tobacco products interfered with osteogenic differentiation of human embryonic stem cells, a well-established surrogate model for human embryonic bone development. Contrasting a diverse array of tobacco products unveiled that sidestream smoke was generally more developmentally osteotoxic than mainstream smoke and that harm-reduction products may not be less harmful than conventional products, adverse effects that were seemingly independent of nicotine.

An Evaluation of Human Induced Pluripotent Stem Cells to Test for Cardiac Developmental Toxicity.

To prevent congenital defects arising from maternal exposure, safety regulations require pre-market developmental toxicity screens for industrial chemicals and pharmaceuticals. Traditional embryotoxicity approaches depend heavily on the use of low-throughput animal models which may not adequately predict human risk. The validated embryonic stem cell test (EST) developed in murine embryonic stem cells addressed the former problem over 15 years ago. Here, we present a proof-of-concept study to address the latter challenge by updating all three endpoints of the classic mouse EST with endpoints derived from human induced pluripotent stem cells (hiPSCs) and human fibroblasts. Exposure of hiPSCs to selected test chemicals inhibited differentiation at lower concentrations than observed in the mouse EST. The hiPSC-EST also discerned adverse developmental outcomes driven by novel environmental toxicants. Evaluation of the early cardiac gene TBX5 yielded similar toxicity patterns as the full-length hiPSC-EST. Together, these findings support the further development of hiPSCs and early molecular endpoints as a biologically relevant embryotoxicity screening approach for individual chemicals and mixtures.

Maternal-to-zygotic transition as a potential target for niclosamide during early embryogenesis.

Niclosamide is an antihelminthic drug used worldwide for the treatment of tapeworm infections. Recent drug repurposing screens have highlighted the broad bioactivity of niclosamide across diverse mechanisms of action. As a result, niclosamide is being evaluated for a range of alternative drug-repurposing applications, including the treatment of cancer, bacterial infections, and Zika virus. As new applications of niclosamide will require non-oral delivery routes that may lead to exposure in utero, it is important to understand the mechanism of niclosamide toxicity during early stages of embryonic development. Previously, we showed that niclosamide induces a concentration-dependent delay in epiboly progression in the absence of effects on oxidative phosphorylation - a well-established target for niclosamide. Therefore, the overall objective of this study was to further examine the mechanism of niclosamide-induced epiboly delay during zebrafish embryogenesis. Based on this study, we found that (1) niclosamide exposure during early zebrafish embryogenesis resulted in a decrease in yolk sac integrity with a concomitant decrease in the presence of yolk sac actin networks and increase in cell size; (2) within whole embryos, niclosamide exposure did not alter non-polar metabolites and lipids, but significantly altered amino acids specific to aminoacyl-tRNA biosynthesis; (3) niclosamide significantly altered transcripts related to translation, transcription, and mRNA processing pathways; and (4) niclosamide did not significantly alter levels of rRNA and tRNA. Overall, our findings suggest that niclosamide may be causing a systemic delay in embryonic development by disrupting the translation of maternally-supplied mRNAs, an effect that may be mediated through disruption of aminoacyl-tRNA biosynthesis.

Video-based kinetic analysis of calcification in live osteogenic human embryonic stem cell cultures reveals the developmentally toxic effect of Snus tobacco extract.

Epidemiological studies suggest tobacco consumption as a probable environmental factor for a variety of congenital anomalies, including low bone mass and increased fracture risk. Despite intensive public health initiatives to publicize the detrimental effects of tobacco use during pregnancy, approximately 10-20% of women in the United States still consume tobacco during pregnancy, some opting for so-called harm-reduction tobacco. These include Snus, a type of orally-consumed yet spit-free chewing tobacco, which is purported to expose users to fewer harmful chemicals. Concerns remain from a developmental health perspective since Snus has not reduced overall health risk to consumers and virtually nothing is known about whether skeletal problems from intrauterine exposure arise in the embryo. Utilizing a newly developed video-based calcification assay we determined that extracts from Snus tobacco hindered calcification of osteoblasts derived from pluripotent stem cells early on in their differentiation. Nicotine, a major component of tobacco products, had no measurable effect in the tested concentration range. However, through the extraction of video data, we determined that the tobacco-specific nitrosamine N'-nitrosonornicotine caused a reduction in calcification with similar kinetics as the complete Snus extract. From measurements of actual nitrosamine concentrations in Snus tobacco extract we furthermore conclude that N'-nitrosonornicotine has the potential to be a major trigger of developmental osteotoxicity caused by Snus tobacco.

Human Pluripotent Stem Cells to Assess Developmental Toxicity in the Osteogenic Lineage.

Musculoskeletal birth defects are frequent, yet their causes remain insufficiently investigated. Aside from genetic factors, exposure to environmental toxicants is suspected to contribute to the etiology of skeletal malformations. However, most chemicals in the environment are insufficiently characterized for their potential to cause harm to the differentiation of osteoblasts, the bone-forming cells and thereby the development of the skeleton.This lack of information primarily stems from animal testing being prohibitively expensive and time-consuming, which has prompted the development of predictive in vitro alternative methods. With the advent of mouse embryonic stem cells, which represent cells with the potential to become any of the 200 cell types in the body, among them osteoblasts, the past 15 years have borne suitable opportunities to assess chemicals in vitro. However, with an increasing understanding of the differences between mouse and human embryonic development, a need for human-specific developmental toxicity testing has risen. This chapter provides a detailed protocol on how to differentiate human embryonic stem cells into the osteogenic lineage, how to assess differentiation inhibition and how to evaluate such findings in relation to the mitochondrial activity of human embryonic stem cells and human fibroblasts, while exposed to a potential toxicant. Together, these endpoints allow for a human-specific screening of developmental toxicity specifically related to the osteogenic lineage.

Low Osteogenic Yield in Human Pluripotent Stem Cells Associates with Differential Neural Crest Promoter Methylation.

Human pluripotent stem cell-derived osteoblasts possess great potential for use in bone disorder elucidation and repair; however, while the general ability of human pluripotent stem cells to differentiate into osteoblasts and lay down bone-specific matrix has been shown, previous studies lack the complete characterization of the process whereby such osteoblasts are derived as well as a comparison between the osteogenic efficiency of multiple cell lines. Here, we compared the osteogenic potential of two human induced pluripotent stem cell lines (RIV9 and RIV4) to human H9 embryonic stem cells. Generally capable of osteogenic differentiation, the overall osteogenic yield was lower in the RIV9 and RIV4 lines and correlated with differential expression of osteocalcin (OCN) in mature cultures and PAX7 and TWIST1 during early differentiation. In the undifferentiated cells, the promoters of the latter two genes were differentially methylated potentially explaining the variation in differentiation efficiency. Furthermore, the expression signatures of selected neural crest and mesodermal genes and proteins suggested that H9 cells preferentially gave rise to neural crest-derived osteoblasts, whereas the osteoblasts in the RIV9 cultures were generated both through a mesodermal and a neural crest route although each at a lower rate. These data suggest that epigenetic dissimilarities between multiple PSC lines may lead to differences in lineage derivation and mineralization. Since osteoblast progenitors from one origin inadequately repair a defect in the other, these data underscore the importance of screening human pluripotent stem cells lines for the identity of the osteoprogenitors they lay down. Stem Cells 2018;36:349-362.

Glucose-Induced Oxidative Stress Reduces Proliferation in Embryonic Stem Cells via FOXO3A/ß-Catenin-Dependent Transcription of p21(cip1).

Embryonic stem cells (ESCs), which are derived from a peri-implantation embryo, are routinely cultured in medium containing diabetic glucose (Glc) concentrations. While pregnancy in women with pre-existing diabetes may result in small embryos, whether such high Glc levels affect ESC growth remains uncovered. We show here that long-term exposure of ESCs to diabetic Glc inhibits their proliferation, thereby mimicking in vivo findings. Molecularly, Glc exposure increased oxidative stress and activated Forkhead box O3a (FOXO3a), promoting increased expression and activity of the ROS-removal enzymes superoxide dismutase and catalase and the cell-cycle inhibitors p21(cip1) and p27(kip1). Diabetic Glc also promoted ß-catenin nuclear localization and the formation of a complex with FOXO3a that localized to the promoters of Sod2, p21(cip1), and potentially p27(kip1). Our results demonstrate an adaptive response to increases in oxidative stress induced by diabetic Glc conditions that promote ROS removal, but also result in a decrease in proliferation.

Wnt5a Supports Osteogenic Lineage Decisions in Embryonic Stem Cells.

The specification of pluripotent stem cells into the bone-forming osteoblasts has been explored in a number of studies. However, the current body of literature has yet to adequately address the role of Wnt glycoproteins in the differentiation of pluripotent stem cells along the osteogenic lineage. During mouse embryonic stem cell (ESC) in vitro osteogenesis, the noncanonical WNT5a is expressed early on. Cells either sorted by their positive WNT5a expression or when supplemented with recombinant WNT5a (rWNT5a) during a 2-day window showed significantly enhanced osteogenic yield. Mechanistically, rWNT5a supplementation upregulated protein kinase C (PKC), calcium/calmodulin-dependent kinase II (CamKII) and c-Jun N-terminal kinase (JNK) activity while antagonizing the key effector of canonical Wnt signaling: ß-catenin. Conversely, when recombinant WNT3a (rWNT3a) or other positive regulators of ß-catenin were employed during this same time window there was a decrease in osteogenic marker expression. However, if rWNT3a was supplemented during a time window following rWNT5a treatment, osteogenic differentiation was enhanced both in murine and human ESCs. Elucidating the role of these WNT ligands in directing the early stages of osteogenesis has the potential to considerably improve tissue engineering protocols and applications for regenerative medicine.

Functional and morphological plasticity of crocodile (Crocodylus porosus) salt glands.

The estuarine crocodile, Crocodylus porosus, inhabits both freshwater and hypersaline waterways and maintains ionic homeostasis by excreting excess sodium and chloride ions via lingual salt glands. In the present study, we sought to investigate the phenotypic plasticity, both morphological and functional, in the lingual salt glands of the estuarine crocodile associated with chronic exposure to freshwater (FW) and saltwater (SW) environments. Examination of haematological parameters indicated that there were no long-term disruptions to ionic homeostasis with prolonged exposure to SW. Maximal secretory rates from the salt glands of SW-acclimated animals (100.8+/-14.7 micromol 100 g(-0.7) body mass h(-1)) were almost three times greater than those of FW-acclimated animals (31.6+/-6.2 micromol 100 g(-0.7) body mass h(-1)). There were no differences in the mass-specific metabolic rate of salt gland tissue slices from FW- and SW-acclimated animals (558.9+/-49.6 and 527.3+/-142.8 microl O(2) g(-1) h(-1), respectively). Stimulation of the tissue slices from SW-acclimated animals by methacholine resulted in a 33% increase in oxygen consumption rate. There was no significant increase in the metabolic rate of tissues from FW-acclimated animals in response to methacholine. Morphologically, the secretory cells from the salt glands of SW-acclimated animals were larger than those of FW-acclimated animals. In addition, there were significantly more mitochondria per unit volume in secretory tissue from SW-acclimated animals. The results from this study demonstrate that the salt glands of C. porosus are phenotypically plastic, both morphologically and functionally and acclimate to changes in environmental salinity.