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

Abstract

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.

Keywords: developmental toxicity; embryonic stem cells; hypomineralization; osteoblasts; oxidative stress; tobacco smoke solution.

Figure 1

Differentiation inhibition caused by harm-reduction tobacco exposure occurred through intermediate levels of reactive oxygen species. (A) Schematic of experimental design and exposure scheme. Created with BioRender.com. (B) Mineralization assay using Arsenazo III to determine osteogenic differentiation efficiency after exposure to previously identified effective doses of Camel cigarette smoke extracts [9] within different time windows. (C) RUNX2 mRNA expression measured with qRT-PCR, normalized to GAPDH and compared to the untreated control, set at 1; n = 3 ± SD. (D) Superoxide anion content measured upon reaction of the cells with luminol and charted as percent of the untreated cultures; d7, n = 3 ± SD. (E) Cells were exposed for seven days, incubated with MitoSOX, photographed and positive cells counted. Only Camel exposure elicited a significant increase specifically in mitochondrial oxidative stress. (F) Calcium deposit was quantified from cultures exposed for 20 days with and without concomitant addition of antioxidants. Effective doses of tobacco smoke solutions and extracts reduced calcification, which was rescued with antioxidant treatment; n = 3 ± SD. * p < 0.05, One-Way ANOVA versus untreated cultures. AA, ascorbic acid; ED, effective dose; GSHOEt, glutathione reduced ethyl ester; MS, mainstream; NED, non-effective dose; RLU, relative light unit; SS, sidestream; UT, untreated solvent control; VitE, Vitamin E.

Figure 2

Medium throughput transcript analysis reveals common and differentially expressed apoptosis-related mRNAs in hESCs exposed to conventional and harm-reduction Camel smoke extracts. (A,B) Heat maps of apoptotic mRNAs de-regulated in tobacco exposed hESCs as measured with the RT² qPCR array for apoptosis. * p < 0.05 vs UT. (C) VENN diagrams showing relations between differentially expressed transcripts in dependence of cigarette smoke extract type (log₂FC > 1; Adj. p-value < 0.05). Venn diagrams were generated with Venny 2.0 [28]. (D) Table identifying the transcripts uniquely regulated by the two cigarette smoke extracts. (E) Volcano Plot for Camel SS ED and Camel Blue SS ED exposed cells. ED, effective dose; FC, fold change; NED, non-effective dose; SS, sidestream; UT, untreated solvent control.

Figure 3

Camel Blue SS elicits a weaker apoptosis response than Camel SS. (A) RT² qPCR array for apoptosis identified distinct expression patterns of various caspase isoforms between Camel and Camel Blue SS smoke exposed cells. n = 3 ± SD. * p < 0.05, two-tailed t-test. ^Δ p < 0.05, two-tailed t-test between Camel SS ED and Camel Blue SS ED. (B) Western blots revealed the differential activation of caspases associated with extrinsic and intrinsic apoptotic pathways. (C) Accordingly, the executioner caspases 3/7 were highly activated in cells exposed to Camel, but only mildly when exposed to Camel Blue. Antioxidant treatment inhibited this activation. Insets show brightfield images of the same field of view. Bar = 100 µM. (C’) The ratio of BCL2 to BAX mRNA expression suggested an anti-apoptotic response in Camel Blue SS cultures. (D) Inhibition of these caspases rescued calcification in cells treated with effective doses of tobacco products; n = 5 ± SD. * p < 0.05, One-Way ANOVA versus untreated cultures. ^Δ p < 0.05, One-Way ANOVA versus ED. (E) Some proapoptotic genes were found upregulated in both Camel and Camel Blue SS cultures. n = 5 ± SD. * p < 0.05, two-tailed t-test. ^Δ p < 0.05, two-tailed t-test between Camel SS ED and Camel Blue SS ED. (F) LIVE/DEAD assay revealed cell death in cells exposed to conventional smoke extracts only. 4i, caspase 4 inhibitor; 8i, caspase 8 inhibitor; 9i, caspase 9 inhibitor; AA, ascorbic acid; ED, effective dose; NED, non-effective dose; SS, sidestream; UT, untreated solvent control.

Figure 4

Reduced viability in hESCs exposed to conventional Camel extract is due to DNA damage. (A) RT² qPCR array for apoptosis found upregulation of genes associated with DNA damage response in Camel SS smoke exposed cells. n = 3 ± SD. * p < 0.05, two-tailed t-test versus UT, ^Δ p < 0.05, two-tailed t-test between Camel SS ED and Camel Blue SS ED. (B) Western blots confirmed ABL1 activation in Camel SS effective doses at the protein level. (C,D) Comet assays confirm DNA damage in response to Camel exposure, which was absent in Camel Blue exposed cells and cells treated with antioxidant. n = 3 ± SD. * p < 0.05, One-Way ANOVA versus untreated cultures. Scale bar = 63×. 9i, caspase 9 inhibitor; AA, ascorbic acid; ED, effective dose; NED, non-effective dose; SS, sidestream; UT, untreated solvent control.

Figure 5

Deterioration of mitochondrial health in exposed hESCs. (A) Mitochondrial membrane potential measurements revealed a reduced membrane potential in Camel exposed cells as a sign for execution of the intrinsic apoptotic pathway. * p < 0.05, One-Way ANOVA versus untreated or NED cultures (B) AMP-to-ATP ratio was increased in Camel SS ED, suggesting mitochondrial dysfunction. * p < 0.05, One-Way ANOVA versus untreated cultures. (C) qPCR array analysis revealed upregulation of mRNAs associated with integral mitochondrial apoptosis in both Camel SS and Camel Blue SS ED. * p < 0.05, two-tailed t-test versus untreated control, ^Δ p < 0.05, two-tailed t-test between Camel SS ED and Camel Blue SS ED. ED, effective dose; NED, non-effective dose; SS, sidestream; UT, untreated solvent control.

Figure 6

Tobacco smoke exposure elicits changes in mitochondrial networks. (A) MitoTracker and MiNA visualization of mitochondrial networks, magnification 63×. (A’) MitoTracker dye analysis revealed increased mitochondrial signal in the Camel Blue SS effective dose. * p < 0.05, One-Way ANOVA versus untreated cultures. (B) Changes to mitochondrial networks were assessed via mean branch length, mitochondrial footprint, and branches per network. * p < 0.05, One-Way ANOVA versus untreated cultures, ^Δ p < 0.05, One-Way ANOVA versus ED, x denotes max or min outliers. 4i, caspase 4 inhibitor; 9i, caspase 9 inhibitor; C, Camel; CB, Camel Blue; ED, effective dose; NED, non-effective dose; SS, sidestream; UT, untreated solvent control.