Perturbations in Osteogenic Cell Fate Following Exposure to Constituents Present in Tobacco: A Combinatorial Study

Abstract

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.

Keywords: cigarettes; developmental toxicity; embryonic stem cells; osteoblasts; smoke solution; tobacco.

Figure 1

Five assays used for generation of ToxPI charts. The table lists the parameters loaded into ToxPI. Each of the five selected biological processes were given their own pie chart slice based off assay data and had equal weight. ToxPI charts were generated for 17 chemicals using 72 in vitro assays across 5 biological processes. The image on the right represents the color code used for each of the processes. Each slice was given its own color.

Figure 2

ToxPI results for 17 HPHCs. (A) Pie charts for 17 of the chemicals on the HPHC list. Refer to Figure 1 for color coding of ToxPI pie slices. (B) ToxPI charts. Benz[a]anthracene (3.717) through N-nitrosodimethylamine (0.102) had designated “hits” and produced positive ToxPi values (0 < X). The remainder of the chemicals, including nicotine, had ToxPI-predicted null effects (X = 0). (B) Toxicity Forecast data averages and in vitro H9 hESC ID₅₀/IC₅₀ µg/mL values obtained from concentration–response curves. The average assay hit value in µM (AC₅₀) for each of the tested ToxPI positive constituents was obtained from the Toxicity Forecaster database. The AC₅₀ in µM for was then converted into µg/mL for appropriate cross-comparison to the H9 hESC ID₅₀/IC₅₀ values calculated from the in vitro osteogenic screen (see page 8).

Figure 3

Differentiation and cell viability endpoints measured for a negative and positive reference chemical. Mitochondrial dehydrogenase activity and calcium content (mg Ca²⁺/mg protein) in response to chemical exposure charted as average ± SD from n = 15 technical replicates representing three biological replicates. hESC, human embryonic stem cell; hFF, human foreskin fibroblast; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylterazolium bromide.

Figure 4

Concentration–response curves. (A) Three ToxPI positive constituents elicited an effect on cytotoxicity and osteogenic differentiation. (B) All of the ToxPI null chemicals caused inhibition at relatively high concentrations. (C) Table listing IC₅₀ and ID₅₀ values, calculated via GraphPad Prism and expressed as µg/mL, embryotoxicity classes calculated according to Genschow et al. [24], and content per cigarette (cig) or gram tobacco as well as actual and calculated plasma concentrations assuming a plasma volume of 3000 mL. Embryotoxicity classes are given in colored circles. ID₅₀ = half-maximal inhibitory concentration of differentiation. IC₅₀ = half-maximal concentration of cytotoxicity. hESC, human embryonic stem cell; hFF, human foreskin fibroblast; ND, not determined from the literature [37,38,39,40,41,42,43,44,45,46,47,48,49].

Figure 5

Skeletal embryotoxicity predictions for additional tobacco smoke constituents with existing ToxCast data. (A) All three constituents were forecasted to elicit embryotoxicity based on ToxPI predictions. (B) Screening with the hESTo classified all three constituents as developmentally osteotoxic. All curves were graphed via GraphPad Prism. Embryotoxicity classes [24] are given in colored circles. hESC, human embryonic stem cell; hFF, human foreskin fibroblast.

Figure 6

hESTo data for additional tobacco smoke constituents with non-existing ToxCast data. Curves were graphed via GraphPad Prism. Embryotoxicity classes [24] are given in colored circles. hESC, human embryonic stem cell; hFF, human foreskin fibroblast.

Figure 7

Double combination heatmaps. MTT (A) and calcium (B) assays corresponding to double combinatorial exposures. In most combinations, cell survival was maintained at or above 100%; however, differentiation was inhibited in almost all combinations. Heatmaps were generated via GraphPad Prism. Data were normalized to solvent control (0.1% DMSO). AA, acetaldehyde; BAP, benzo[a]pyrene; CAT, catechol; COU, coumarin; NIC, nicotine; Q, quinoline; A, acrolein.

Figure 8

Heatmaps pertaining to triple exposure combinations. (A,B) Triple combination heatmaps. MTT (A) and calcium (B) assays corresponding to triple combinatorial exposures. Heatmaps were generated via GraphPad Prism. In most combinations, the addition of acrolein (in comparison to double combinations) maintained cell survival at or above 100%. Data were normalized to solvent control (0.1% DMSO). (C) Calcium content was set in relation to MTT results and a clustered heatmap was generated from the resulting ratios using ClustVis [36]. AA, acetaldehyde; BAP, benzo[a]pyrene; CAT, catechol; COU, coumarin; NIC, nicotine; Q, quinoline; A, acrolein.