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In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures.

Schlage WK, Iskandar AR, Kostadinova R, Xiang Y, Sewer A, Majeed S, Kuehn D, Frentzel S, Talikka M, Geertz M, Mathis C, Ivanov N, Hoeng J, Peitsch MC - Toxicol. Mech. Methods (2014)

Bottom Line: CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues.Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset.These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products.

View Article: PubMed Central - PubMed

Affiliation: Philip Morris International R&D, Philip Morris Products S.A. , Neuchâtel , Switzerland.

ABSTRACT
Smoking has been associated with diseases of the lung, pulmonary airways and oral cavity. Cytologic, genomic and transcriptomic changes in oral mucosa correlate with oral pre-neoplasia, cancer and inflammation (e.g. periodontitis). Alteration of smoking-related gene expression changes in oral epithelial cells is similar to that in bronchial and nasal epithelial cells. Using a systems toxicology approach, we have previously assessed the impact of cigarette smoke (CS) seen as perturbations of biological processes in human nasal and bronchial organotypic epithelial culture models. Here, we report our further assessment using in vitro human oral organotypic epithelium models. We exposed the buccal and gingival organotypic epithelial tissue cultures to CS at the air-liquid interface. CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues. Using microarray technology, gene-set analysis and a novel computational modeling approach leveraging causal biological network models, we identified CS impact on xenobiotic metabolism-related pathways accompanied by a more subtle alteration in inflammatory processes. Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset. These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products.

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CYP1A1/1B1 enzyme activity and cytokine secretion in buccal and gingival tissues. Activity levels of CYP1A1/1B1 were measured at 24 h post-exposure to CS (17.9 and 40.7%) in the buccal (A) and gingival (B) tissues (N = 3 inserts following a single exposure run). Positive control tests are shown (right side of each panel) using TCDD as an inducer of CYP1A1/1B1 activity. Inflammatory mediators (C) and the corresponding gene expression (D) were measured at 24 h post-exposure of CS compared with the air-exposed tissues (N = 3 inserts following a single exposure run). Columns are representing different tissues as indicated in the label under the heatmap. Fold changes were obtained by taking the log2 ratio of the cytokine abundance (C) or of the gene expression (D) between the CS group and air-exposed exposure group for every tissue. Welch’s t-test was performed to test the  hypothesis that cytokine abundance (C) or log2-based gene expression (D) in the CS (19.7 and 40.7%) groups and air-exposed group was equivalent. Fold change was set to be zero for the p > 0.05. Blue and red colors indicate negative or positive fold-changes in the CS-exposed tissues as compared to the air-exposed tissues, respectively. Abbreviations: CCL, CC chemokine ligand; CS, cigarette smoke; CYP, cytochrome; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; IL, interleukin; IP-10, interferon gamma inducible protein 10; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase; SEM, standard error of the mean; RANTES, regulated on activation, normal T cell expressed and secreted; RLU, raw luminescence unit; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; VEGF, vascular endothelial growth factor; PE, post-exposure.
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f8: CYP1A1/1B1 enzyme activity and cytokine secretion in buccal and gingival tissues. Activity levels of CYP1A1/1B1 were measured at 24 h post-exposure to CS (17.9 and 40.7%) in the buccal (A) and gingival (B) tissues (N = 3 inserts following a single exposure run). Positive control tests are shown (right side of each panel) using TCDD as an inducer of CYP1A1/1B1 activity. Inflammatory mediators (C) and the corresponding gene expression (D) were measured at 24 h post-exposure of CS compared with the air-exposed tissues (N = 3 inserts following a single exposure run). Columns are representing different tissues as indicated in the label under the heatmap. Fold changes were obtained by taking the log2 ratio of the cytokine abundance (C) or of the gene expression (D) between the CS group and air-exposed exposure group for every tissue. Welch’s t-test was performed to test the hypothesis that cytokine abundance (C) or log2-based gene expression (D) in the CS (19.7 and 40.7%) groups and air-exposed group was equivalent. Fold change was set to be zero for the p > 0.05. Blue and red colors indicate negative or positive fold-changes in the CS-exposed tissues as compared to the air-exposed tissues, respectively. Abbreviations: CCL, CC chemokine ligand; CS, cigarette smoke; CYP, cytochrome; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; IL, interleukin; IP-10, interferon gamma inducible protein 10; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase; SEM, standard error of the mean; RANTES, regulated on activation, normal T cell expressed and secreted; RLU, raw luminescence unit; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; VEGF, vascular endothelial growth factor; PE, post-exposure.

Mentions: Both buccal and gingival tissues had basal activities of CYP1A1/1B1 enzyme that were measured at 48 h post-exposure (Figure 8A and B). CYP1A1 and CYP1B1 have been shown to metabolize tobacco smoke constituents (Port et al., 2004). Tissues exposed to 19.7% CS, had increased levels of CYP1A1/1B1 activity, although the increase in the buccal tissues could not reach statistical significance (Figure 8A and B). The activities of the CYP1A1/1B1 were not altered in both tissues exposed to the higher concentration of CS as compared to the air-exposed tissues.Figure 8.


In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures.

Schlage WK, Iskandar AR, Kostadinova R, Xiang Y, Sewer A, Majeed S, Kuehn D, Frentzel S, Talikka M, Geertz M, Mathis C, Ivanov N, Hoeng J, Peitsch MC - Toxicol. Mech. Methods (2014)

CYP1A1/1B1 enzyme activity and cytokine secretion in buccal and gingival tissues. Activity levels of CYP1A1/1B1 were measured at 24 h post-exposure to CS (17.9 and 40.7%) in the buccal (A) and gingival (B) tissues (N = 3 inserts following a single exposure run). Positive control tests are shown (right side of each panel) using TCDD as an inducer of CYP1A1/1B1 activity. Inflammatory mediators (C) and the corresponding gene expression (D) were measured at 24 h post-exposure of CS compared with the air-exposed tissues (N = 3 inserts following a single exposure run). Columns are representing different tissues as indicated in the label under the heatmap. Fold changes were obtained by taking the log2 ratio of the cytokine abundance (C) or of the gene expression (D) between the CS group and air-exposed exposure group for every tissue. Welch’s t-test was performed to test the  hypothesis that cytokine abundance (C) or log2-based gene expression (D) in the CS (19.7 and 40.7%) groups and air-exposed group was equivalent. Fold change was set to be zero for the p > 0.05. Blue and red colors indicate negative or positive fold-changes in the CS-exposed tissues as compared to the air-exposed tissues, respectively. Abbreviations: CCL, CC chemokine ligand; CS, cigarette smoke; CYP, cytochrome; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; IL, interleukin; IP-10, interferon gamma inducible protein 10; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase; SEM, standard error of the mean; RANTES, regulated on activation, normal T cell expressed and secreted; RLU, raw luminescence unit; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; VEGF, vascular endothelial growth factor; PE, post-exposure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219813&req=5

f8: CYP1A1/1B1 enzyme activity and cytokine secretion in buccal and gingival tissues. Activity levels of CYP1A1/1B1 were measured at 24 h post-exposure to CS (17.9 and 40.7%) in the buccal (A) and gingival (B) tissues (N = 3 inserts following a single exposure run). Positive control tests are shown (right side of each panel) using TCDD as an inducer of CYP1A1/1B1 activity. Inflammatory mediators (C) and the corresponding gene expression (D) were measured at 24 h post-exposure of CS compared with the air-exposed tissues (N = 3 inserts following a single exposure run). Columns are representing different tissues as indicated in the label under the heatmap. Fold changes were obtained by taking the log2 ratio of the cytokine abundance (C) or of the gene expression (D) between the CS group and air-exposed exposure group for every tissue. Welch’s t-test was performed to test the hypothesis that cytokine abundance (C) or log2-based gene expression (D) in the CS (19.7 and 40.7%) groups and air-exposed group was equivalent. Fold change was set to be zero for the p > 0.05. Blue and red colors indicate negative or positive fold-changes in the CS-exposed tissues as compared to the air-exposed tissues, respectively. Abbreviations: CCL, CC chemokine ligand; CS, cigarette smoke; CYP, cytochrome; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; IL, interleukin; IP-10, interferon gamma inducible protein 10; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase; SEM, standard error of the mean; RANTES, regulated on activation, normal T cell expressed and secreted; RLU, raw luminescence unit; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; VEGF, vascular endothelial growth factor; PE, post-exposure.
Mentions: Both buccal and gingival tissues had basal activities of CYP1A1/1B1 enzyme that were measured at 48 h post-exposure (Figure 8A and B). CYP1A1 and CYP1B1 have been shown to metabolize tobacco smoke constituents (Port et al., 2004). Tissues exposed to 19.7% CS, had increased levels of CYP1A1/1B1 activity, although the increase in the buccal tissues could not reach statistical significance (Figure 8A and B). The activities of the CYP1A1/1B1 were not altered in both tissues exposed to the higher concentration of CS as compared to the air-exposed tissues.Figure 8.

Bottom Line: CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues.Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset.These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products.

View Article: PubMed Central - PubMed

Affiliation: Philip Morris International R&D, Philip Morris Products S.A. , Neuchâtel , Switzerland.

ABSTRACT
Smoking has been associated with diseases of the lung, pulmonary airways and oral cavity. Cytologic, genomic and transcriptomic changes in oral mucosa correlate with oral pre-neoplasia, cancer and inflammation (e.g. periodontitis). Alteration of smoking-related gene expression changes in oral epithelial cells is similar to that in bronchial and nasal epithelial cells. Using a systems toxicology approach, we have previously assessed the impact of cigarette smoke (CS) seen as perturbations of biological processes in human nasal and bronchial organotypic epithelial culture models. Here, we report our further assessment using in vitro human oral organotypic epithelium models. We exposed the buccal and gingival organotypic epithelial tissue cultures to CS at the air-liquid interface. CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues. Using microarray technology, gene-set analysis and a novel computational modeling approach leveraging causal biological network models, we identified CS impact on xenobiotic metabolism-related pathways accompanied by a more subtle alteration in inflammatory processes. Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset. These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products.

Show MeSH
Related in: MedlinePlus