RESUMEN

Tobacco smoke contains between 7000 and 10,000 constituents, and only an evanescently low number of which have been identified, let alone been evaluated for their toxicity. Recently, the Food and Drug Administration has published a list of 93 chemical tobacco constituents that are harmful or potentially harmful to a number of cellular processes. However, their effect on developing skeletal cells is unknown. In this study, we used ToxPI, a computational tool, to prioritize constituents on this list for screening in osteogenically differentiating human embryonic stem cells and fibroblasts. In selected endpoint assays, we evaluated the potential of these chemicals to inhibit osteogenic differentiation success as well as their cytotoxicity. Six of these chemicals, which were ascribed an embryotoxic potential in our screen, as well as nicotine, which was not found to be osteotoxic in vitro, were then evaluated in combinatorial exposures, either in pairs of two or three. No one single chemical could be pinpointed as the culprit of reduced calcification in response to tobacco exposure. Combining chemicals at their half-maximal inhibitory concentration of differentiation often elicited expected decreases in calcification over the individual exposures; however, cytotoxicity was improved in many of the dual combinations. A reverse response was also noted, in which calcification output improved in combinatorial exposures. Results from ternary combinations reflected those from double combinations. Thus, the results from this study suggest that it may be difficult to isolate single chemicals as the primary drivers of skeletal embryotoxicity and that the full combination of chemicals in tobacco smoke may produce the hypomineralization phenotype that we have so far observed in vitro in human embryonic stem cells as well as in vivo in zebrafish.

RESUMEN

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.

RESUMEN

Exposure to cigarette smoke represents the largest source of preventable death and disease in the United States. This may be in part due to the nature of the delayed harmful effects as well as the lack of awareness of the scope of harm presented by these products. The presence of "light" versions further clouds the harmful effects of tobacco products. While active smoking in expectant mothers may be reduced by educational and outreach campaigns, exposure to secondhand smoke is often involuntary yet may harm the developing embryo. In this study, we show that the main component of secondhand smoke, sidestream cigarette smoke, from several brands, including harm-reduction versions, triggered unsuccessful hatching at 3 dpf and reduced overall survival at 6 dpf in developing zebrafish. At non-lethal concentrations, craniofacial defects with different severity based on the cigarette smoke extract were noted by 6 dpf. All tested products, including harm-reduction products, significantly impacted cartilage formation and/or bone mineralization in zebrafish embryos, independent of whether the bones/cartilage formed from the mesoderm or neural crest. Together, these results in a model system often used to detect embryonic malformations imply that exposure of a woman to secondhand smoke while pregnant may lead to mineralization issues in the skeleton of her newborn, ultimately adding a direct in utero association to the increased fracture risk observed in children of mothers exposed to cigarette smoke.

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RESUMEN

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.

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RESUMEN

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.

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