Wanda R. Fields

Matrix-degrading and pro-inflammatory changes in human vascular endothelial cells exposed to cigarette smoke condensate

Cigarette smoking has been associated with an increase in the severity and prevalence of atherosclerosis in the abdominal aorta. To begin our investigation ofthis finding, we used an integrated approach combining gene expression profiling, protein analysis, cytokine measurements, and cytotoxicity determinations to examine molecular responses of cultured human aortic and coronary endothelial cells exposed to cigarette smoke condensate (CSC) and nicotine. Exposure of endothelial cells to CSC (30 and 60 μg/mL TPM) for 24 h resulted in minimal cytotoxicity, and the upregulation of genes involved in matrix degradation (MMP-1, MMP-8, and MMP-9), xenobiotic metabolism (HO-1 and CYP1A2), and downregulation of genes involved in cell cycle regulation (including TOP2A, CCNB1, CCNA, CDKN3). Exposure of cells to a high physiological concentration of nicotine resulted in few differentially expressed genes. Immunoblot analysis of proteins selected from genes shown to be differentially regulated by microarray analysis revealed similar responses. Finally, a number of inflammatory cytokines measured in culture media were elevated in response to CSC. Together, these results describe a complex proinflammatory response, possibly mediating the recruitment of leukocytes through cytokine signaling. Additionally, fibrous cap destabilization may be facilitated by matrix metalloproteinase upregulation.

CHEMOPROTECTIVE FUNCTIONS OF GLUTATHIONE S-TRANSFERASES IN CELL LINES INDUCED TO EXPRESS SPECIFIC ISOZYMES BY STABLE TRANSFECTION

The authors have shown that expression of mGSTM1-1 or hGSTP1-1 in MCF-7 cells protects against DNA alkylation by 4-nitroquinoline-1-oxide (NQO) in an isozyme-specific manner and is commensurate with relative specific activity. Expression of GSTs also conferred protection against both DNA strand breaks and sister-chromatid exchange induced by NQO. Interestingly, GST expression did not protect against NQO cytotoxicity in transfected MCF-7 cell lines, although resistance to NQO cytotoxicity was observed in a T47D pi transfectant line, expressing much higher specific activity of the transfected hGSTP1-1. However, high level expression of hGSTP1-1 or mGSTM1-1 in V79 transfectants did not confer resistance to cytotoxicity, indicating that expression of GST alone is not sufficient. The authors have also shown protection against AFB1 in cell lines expressing transfected rat CYP2B1 (V79MZr2B1) and transfected mGST-Yc (mGSTA3-3). Protection was observed against both alkylation of DNA (3-fold) by [3H]AFB1 and against AFB1 cytotoxicity (7-fold). Similarly, V79MZr1A1 cells that express CYP1A1 and either transfected human or murine GSTP1-1 (< 5000 mIU/mg, CDNB) exhibited > 70% decrease in covalent labeling of total nucleic acids by [3H]BPDE. However, no protection against the cytotoxicity of BPDE was conferred by expression of hGSTP1-1. Overall, these results indicate that in some (NQO or BPDE), but not all (AFB1) cases, protection by GST expression against DNA damage is more effective than protection against cytotoxicity. In addition, there is evidence to indicate that additional factor(s) other than high GST isozyme expression level and good substrate efficacy affect the degree of protection against cytotoxicity of reactive electrophiles. This includes the differential protection against NQO cytotoxicity in T47D pi, but not V79 Xh pi-33 cells and also the recent studies which showed that expression of the MRP GS-X conjugate efflux transporter confers synergistic protection against NQO cytotoxicity when co-expressed with transfected human GSTP1-1 in MCF-7 cells. Thus, protective efficacy conferred by GST expression can vary with different cellular targets and/or experimental end-points, as well as with variations in relative specific activity or in different cellular phenotypic contexts.

Distinct regulatory profiles of interleukins and chemokines in response to cigarette smoke condensate in normal human bronchial epithelial (NHBE) cells.

Bronchial epithelium is frequently exposed to air pollutants, and it is hypothesized that these cells elicit inflammatory responses as early elements in pulmonary defense. Our purpose was to evaluate changes in messenger RNA levels of 84 genes representing cytokines and receptors over a repetitive-exposure time course to further define the inflammatory responses associated with mainstream cigarette smoke (MSS) exposure in an in vitro lung model. Normal human bronchial epithelial cells were treated with mainstream cigarette smoke condensate (CSC) prepared from Kentucky 2R4F cigarettes (60 microg total particulate matter/mL media, 0.2% dimethylsulfoxide), and examined by quantitative real-time polymerase chain reaction. Applications of CSC were designed in seven groups to test immediate, early, intermediate, and late responses evaluated at the end of alternating exposure/recovery periods. Three predominant gene expression responses were observed: adaptive (return to baseline), sustained (maintained expression during treatment), and chronic (maintained expression posttreatment). Overall, 25 genes exhibited statistically significant changes: 14 genes exclusively elevated, 10 genes exclusively depressed, and 1, interleukin-8 (IL8), exhibiting both up- and downregulation in the seven groups. The most responsive genes were osteopontin (34-fold upregulation) and CXCL14 (23-fold downregulation). Our observations suggest that specific genes involved in inflammatory pathways respond to CSC in chronic, sustained, or adaptive patterns with the chronic pattern as the predominant behavior.

The time course of expression of genes involved in specific pathways in normal human bronchial epithelial cells following exposure to cigarette smoke.

This study was conducted to determine the time course of gene expression associated with specific signaling pathways in normal human bronchial epithelial (NHBE) cells after exposure to 2 concentrations of 2R4F tobacco mainstream smoke (MSS). Expression of 84 genes representing 18 signal transduction pathways was quantitated in MSS- and air-exposed cultures using real-time polymerase chain reaction (PCR) arrays at 1, 4, and 24 hours following exposure. A confidence score, calculated based on statistical analysis of the degree and reproducibility of expression changes, was used to identify potential biologically significant changes in gene expression. Stimulation of NIAP, an apoptosis inhibitor, suppression of NFKB1 and MYC, representing pro-apoptotic activity, and down-regulation of TCF7 and up-regulation of KLK2, representing anti-/pro-inflammatory responses, were altered 1 hour after exposure to the high concentration of MSS. At the 4-hour time point, the pattern had changed such that 10 different genes were now up-regulated and an additional gene was now down-regulated. Significant changes included genes involved in inflammatory response (LTA, SELPLG, and IL8), repair and wound-healing activity (MMP10), and growth activity (GREB1, EGR1), suggesting repair in this period. By 24 hours, the only up-regulated genes in common with the 4-hour profile were SELPLG and IL8, suggesting continued inflammatory signaling. These results suggest that identification of specific gene expression–based biomarkers of MSS toxicity is promising for investigating specific mechanisms of cellular damage. As expected, the expressed signals were dependent on the concentration of MSS and the postexposure times.

Development, qualification, validation and application of the Ames test using a VITROCELL® VC10® smoke exposure system

Graphical abstract

Abstract 4980: Comparison of gene expression profiles of cigarette smoke-exposed normal human bronchial epithelial (NHBE) to profiles from smokers and patients with chronic obstructive pulmonary disease (COPD) and lung cancer

Molecular changes in multiple signaling pathways that regulate oxidative stress, inflammation and xenobiotic metabolism are frequently observed in diseased lung. The purposes of this study were: 1) to assess differential gene expression profiles in human lung tissue or cells of non-smokers and smokers with and without lung cancer or COPD and 2) to compare gene expression changes induced by smoke in in vitro cultures of NHBE cells to expression profiles from ex vivo lung tissue samples. Gene expression profiles were evaluated with the Affymetrix HG_U133 Plus 2.0 GeneChip Microarray. First, expression profiles which distinguished normal tissues from non-smokers and smokers were represented by 115 statistically significant probe sets (genes). Pathway and gene ontology analyses identified several ontologies containing overrepresented genes in smokers, including those involved in immune response and T cell activation. Second, gene profiles (7880 probe sets) and molecular pathways that distinguished malignant lung of smokers from the paired normal tissue were associated with immune response, cell cycle regulation and tryptophan metabolism. Third, comparisons of bronchial brushings from non-smoker, smoker and COPD (GOLD 0 – II) yielded 63 unique probe sets. NADPH quinone oxidoreductase (NQO1), mucin 5AC and glutamate decarboxylase 1 were among notable targets, and the top pathways represented oxidative stress, xenobiotic metabolism and hypoxia. Finally, as we previously reported, the top responders in the NHBE cells exposed to repeated applications of cigarette smoke condensate (CSC) were represented by 241 consistently regulated probe sets with significant deregulation of genes involved in oxidative stress, xenobiotic and tryptophan metabolism. Bioinformatic analyses were further employed to identify correlations with smoke and disease-impact in ex vivo lung samples. The gene profiles that were significantly modified in both CSC-exposed NHBE cells and COPD lung brushings included aldo-keto reductases (AKR1B10, AKR1C1, AKR1C2), cytochrome P450 1B1 (CYP1B1), NQO1, glutathione peroxidase 2 (GPX2) and SLC7A11, an amino acid transporter. Probe sets significantly regulated in both CSC-exposed NHBE cells and the cancerous and smoking-exposed lung were osteopontin (OPN/Spp1), dedicator of cytokinesis 10 (DOCK10), glycerol kinase (GK), kynureninase (KYNU) and transforming growth factor beta 2 (TGFB2). Collectively, the data suggest that smoke-induced expression profiles in in vitro lung cell models are phenotypically relevant to lung modifications in vivo and may serve as potential biomarkers of effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4980. doi:10.1158/1538-7445.AM2011-4980

Abstract 1693: Assessment of Nrf2 luciferase reporter assay and heme oxygenase expression as models of oxidative stress in in vitro cultures of lung cells exposed to cigarette smoke condensate

The Nrf2 signaling pathway is an important regulator associated with oxidative stress, xenobiotic metabolism, DNA damage and anti-inflammatory response. A key responsive gene in the Nrf2 regulatory pathway is heme oxygense 1 (HO-1), a cytoprotective enzyme that serves in an antioxidant capacity. An imbalance in antioxidant capacity observed in animal models or individuals with specific Nrf2 and HO-1 polymorphisms has been associated with susceptibility to lung diseases. The objective of this study was to characterize the response of Nrf 2 promoter regulation and HO-1 expression following exposure to cigarette smoke in lung models for oxidative stress. Cigarette smoke condensate (CSC) was prepared from Kentucky 3R4F (K3R4F) reference cigarettes. Nrf2 promoter regulation and HO-1 expression were evaluated in lung cells exposed to K3R4F CSC (0 − 90μg total particulate matter/ml media). Nrf2 promoter regulation was evaluated in transiently-transfected Nrf2 luciferase reporter cells exposed to K3R4F CSC for 0.5, 1, 2, 6 or 24 hours followed by quantification of luciferase activity. HO-1 expression was assessed following exposure to the K3R4F CSC in normal human bronchial epithelial (NHBE) cells for 3, 6 or 24 hours using real-time quantitative RT/PCR and enzyme linked immunoassay (ELISA). Nrf2 reporter cells exhibited dose-dependent increases in promoter activation following CSC exposure. The maximum response was observed between 6 and 24 hours of exposure with 15-, 25-, and 40-fold increases across the exposure range. The specificity of the promoter response was confirmed with co- exposure of the inhibitor all-trans retinoic acid with CSC. The CSC-induced Nrf2 activation was reduced towards control levels in the presence of the inhibitor. CSC also induced dose- and time-dependent increases on HO-1 mRNA and protein levels in NHBE cells. The mRNA changes ranged from 10- to 70-fold with maximal responses observed between 3 and 6 hours. The dynamic range of HO-1 protein response was observed and reached a level of 11-fold increase by 60 μg/ml. Collectively, these responses are consistent with our previously reported study which identified oxidative stress responsive genes in NHBE cells exposed to repetitive applications of CSC. The data suggest that changes in the Nrf2 regulatory pathway may serve as an effective indicator of oxidative stress and the impact of tobacco products in in vitro lung cell culture models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1693.