Part three: a randomized study to assess biomarker changes in cigarette smokers switched to Vuse Solo or Abstinence.

Biomarkers of exposure (BoE) can help evaluate exposure to combustion-related, tobacco-specific toxicants after smokers switch from cigarettes to potentially less-harmful products like electronic nicotine delivery systems (ENDS). This paper reports data for one (Vuse Solo Original) of three products evaluated in a randomized, controlled, confinement study of BoE in smokers switched to ENDS. Subjects smoked their usual brand cigarette ad libitum for two days, then were randomized to one of three ENDS for a 7-day ad libitum use period, or to smoking abstinence. Thirteen BoE were assessed at baseline and Day 5, and percent change in mean values for each BoE was calculated. Biomarkers of potential harm (BoPH) linked to oxidative stress, platelet activation, and inflammation were also assessed. Levels decreased among subjects randomized to Vuse Solo versus Abstinence, respectively, for the following BoE: 42-96% versus 52-97% (non-nicotine constituents); 51% versus 55% (blood carboxyhemoglobin); and 29% versus 96% (nicotine exposure). Significant decreases were observed in three BoPH: leukotriene E4, 11-dehydro-thromboxane B2, and 2,3-dinor thromboxane B2 on Day 7 in the Vuse Solo and Abstinence groups. These findings show that ENDS use results in substantially reduced exposure to toxicants compared to smoking, which may lead to reduced biological effects.

Differential gene expression of 3D primary human airway cultures exposed to cigarette smoke and electronic nicotine delivery system (ENDS) preparations.

BACKGROUND: Acute exposure to cigarette smoke alters gene expression in several biological pathways such as apoptosis, immune response, tumorigenesis and stress response, among others. However, the effects of electronic nicotine delivery systems (ENDS) on early changes in gene expression is relatively unknown. The objective of this study was to evaluate the early toxicogenomic changes using a fully-differentiated primary normal human bronchial epithelial (NHBE) culture model after an acute exposure to cigarette and ENDS preparations. RESULTS: RNA sequencing and pathway enrichment analysis identified time and dose dependent changes in gene expression and several canonical pathways when exposed to cigarette preparations compared to vehicle control, including oxidative stress, xenobiotic metabolism, SPINK1 general cancer pathways and mucociliary clearance. No changes were observed with ENDS preparations containing up to 28 µg/mL nicotine. Full model hierarchical clustering revealed that ENDS preparations were similar to vehicle control. CONCLUSION: This study revealed that while an acute exposure to cigarette preparations significantly and differentially regulated many genes and canonical pathways, ENDS preparations containing the same concentration of nicotine had very little effect on gene expression in fully-differentiated primary NHBE cultures.

Transcriptomic Response of Primary Human Bronchial Cells to Repeated Exposures of Cigarette and ENDS Preparations.

Cigarette smoke deregulates several biological pathways by modulating gene expression in airway epithelial cells and altering the physiology of the airway epithelium. The effects of repeated exposures of electronic cigarette delivery systems (ENDS) on gene expression in airway epithelium are relatively unknown. In order to assess the effect of repeated exposures of ENDS, primary normal human bronchial epithelial (NHBE) cells grown at air-liquid interface (ALI) were exposed to cigarette and ENDS preparations daily for 10 days. Cigarette smoke preparations significantly altered gene expression in a dose-dependent manner compared to vehicle control, including genes linked to oxidative stress, xenobiotic metabolism, cancer pathways, epithelial-mesenchymal transition, fatty acid metabolism, degradation of collagen and extracellular matrix, O-glycosylation, and chemokines/cytokines, which are known pathways found to be altered in smokers. Conversely, ENDS preparations had minimal effect on transcriptional pathways. This study revealed that a sub-chronic exposure of primary NHBE cultures to cigarette and ENDS preparations differentially regulated genes and canonical pathways, with minimal effect observed with ENDS preparations compared to cigarette preparations. This study also demonstrates the versatility of primary NHBE cultures at ALI to evaluate repeat-dose exposures of tobacco products.

Cigarette smoke preparations, not electronic nicotine delivery system preparations, induce features of lung disease in a 3D lung repeat-dose model.

Cigarette smoking is a risk factor for several lung diseases, including chronic obstructive pulmonary disease, cardiovascular disease, and lung cancer. The potential health effects of chronic use of electronic nicotine delivery systems (ENDS) is unclear. This study utilized fully differentiated primary normal human bronchial epithelial (NHBE) cultures in a repeat-dose exposure to evaluate and compare the effect of combustible cigarette and ENDS preparations. We show that 1-h daily exposure of NHBE cultures over a 10-day period to combustible cigarette whole smoke-conditioned media (WS-CM) increased expression of oxidative stress markers, cell proliferation, airway remodeling, and cellular transformation markers and decreased mucociliary function including ion channel function and airway surface liquid. Conversely, aerosol conditioned media (ACM) from ENDS with similar nicotine concentration (equivalent-nicotine units) as WS-CM and nicotine alone had no effect on those parameters. In conclusion, primary NHBE cultures in a repeat-dose exposure system represent a good model to assess the features of lung disease. This study also reveals that cigarette and ENDS preparations differentially elicit several key endpoints, some of which are potential biomarkers for lung cancer or chronic obstructive pulmonary disease (COPD).

Metabolomic Analysis Identified Reduced Levels of Xenobiotics, Oxidative Stress, and Improved Vitamin Metabolism in Smokers Switched to Vuse Electronic Nicotine Delivery System.

INTRODUCTION: Switching to noncombustible tobacco products presents an opportunity for cigarette smokers to potentially reduce the health risks associated with smoking. Electronic Nicotine Delivery Systems (ENDS) are one such product because the vapor produced from ENDS contains far fewer toxicants than cigarette smoke. To investigate the biochemical effects of switching from smoking to an ENDS, we assessed global metabolomic profiles of smokers in a 7-day confinement clinical study. METHODS: In the first 2 days of this clinical study, the subjects used their usual brand of cigarettes and then switched to exclusive ENDS ad libitum use for 5 days. Urine and plasma samples were collected at baseline and 5 days after switching. The samples were analyzed using a mass spectrometry-based metabolomic platform. RESULTS: Random forest analyses of urine and plasma metabolomic data revealed excellent predictive accuracy (>97%) of a 30-metabolite signature that can differentiate smokers from 5-day ENDS switchers. In these signatures, most biomarkers are nicotine-derived metabolites or xenobiotics. They were significantly reduced in urine and plasma, suggesting a decreased xenobiotic load on subjects. Our results also show significantly decreased levels of plasma glutathione metabolites after switching, which suggests reduced levels of oxidative stress. In addition, increased urinary and plasma levels of vitamins and antioxidants were identified, suggesting enhanced bioavailability due to discontinuation of cigarette smoking and switching to Vuse ENDS use. CONCLUSIONS: Our results suggest reduced toxicant exposure, reduced oxidative stress, and potential beneficial changes in vitamin metabolism within 5 days in smokers switching to Vuse ENDS. IMPLICATIONS: Switching from smoking to exclusive ENDS use in clinical confinement settings results in significant reduction of nicotine metabolites and other cigarette-related xenobiotics in urine and plasma of subjects. Significantly decreased oxidative stress-related metabolites and increased urinary and plasma levels of vitamin metabolites and antioxidants in 5-day short-term ENDS switchers suggest less toxic physiological environment for consumers of ENDS products and potential health benefits if such changes persist.

Differences in biomarkers of inflammation and immune responses in chronic smokers and moist snuff users.

BACKGROUND: Cigarette smoking is a major risk factor for cancer and other diseases. While smoking induces chronic inflammation and aberrant immune responses, the effects of smokeless tobacco products (STPs) on immune responses is less clear. Here we evaluated markers related to immune regulation in smokers (SMK), moist snuff consumers (MSC) and non-tobacco consumers (NTC) to better understand the effects of chronic tobacco use. MATERIALS AND METHODS: Several markers associated with immune regulation were measured in peripheral blood mononuclear cells (PBMCs) from SMK (n = 40), MSC (n = 40), and NTC (n = 40) by flow cytometry. RESULTS: Relative to NTC, seven markers were significantly suppressed in SMK, whereas in MSC, only one marker was significantly suppressed. In a logistic regression model, markers including granzyme B+ lymphocytes, perforin+ lymphocytes, granzyme B+ CD8+T cells, and KLRB1+ CD8+ T cells remained as statistically significant predictors for classifying the three cohorts. Further, cell-surface receptor signaling pathways and cell-cell signaling processes were downregulated in SMK relative to MSC; chemotaxis and LPS-mediated signaling pathways, were upregulated in SMK compared to MSC. A network of the tested markers was constructed to visualize the immunosuppression in SMK relative to MSC. CONCLUSION: Moist snuff consumption is associated with significantly fewer perturbations in inflammation and immune function biomarkers relative to smoking. IMPACT: This work identifies several key immunological biomarkers that differentiate the effects of chronic smoking from the use of moist snuff. Additionally, a molecular basis for aberrant immune responses that could render smokers more susceptible for infections and cancer is provided.

Pathway Analysis of Global Metabolomic Profiles Identified Enrichment of Caffeine, Energy, and Arginine Metabolism in Smokers but Not Moist Snuff Consumers.

Existing US epidemiological data demonstrate that consumption of smokeless tobacco, particularly moist snuff, is less harmful than cigarette smoking. However, the molecular and biochemical changes due to moist snuff consumption relative to smoking remain incompletely understood. We previously reported that smokers (SMK) exhibit elevated oxidative stress and inflammation relative to moist snuff consumers (MSC) and non-tobacco consumers (NTC), based on metabolomic profiling data of saliva, plasma, and urine from MSC, SMK, and NTC. In this study, we investigated the effects of tobacco consumption on additional metabolic pathways using pathway-based analysis tools. To this end, metabolic pathway enrichment analysis and topology analysis were performed through pair-wise comparisons of global metabolomic profiles of SMK, MSC, and NTC. The analyses identified >8 significantly perturbed metabolic pathways in SMK compared with NTC and MSC in all 3 matrices. Among these differentially enriched pathways, perturbations of caffeine metabolism, energy metabolism, and arginine metabolism were mostly observed. In comparison, fewer enriched metabolic pathways were identified in MSC compared with NTC (5 in plasma, none in urine and saliva). This is consistent with our transcriptomics profiling results that show no significant differences in peripheral blood mononuclear cell gene expression between MSC and NTC. These findings, taken together with our previous biochemical, metabolomic, and transcriptomic analysis results, provide a better understanding of the relative changes in healthy tobacco consumers, and demonstrate that chronic cigarette smoking, relative to the use of smokeless tobacco, results in more pronounced biological changes, which could culminate in smoking-related diseases.

Cigarette smoke preparations, not moist snuff, impair expression of genes involved in immune signaling and cytolytic functions.

Cigarette smoke-induced chronic inflammation is associated with compromised immune responses. To understand how tobacco products impact immune responses, we assessed transcriptomic profiles in peripheral blood mononuclear cells (PBMCs) pretreated with Whole Smoke-Conditioned Medium (WS-CM) or Smokeless Tobacco Extracts (STE), and stimulated with lipopolysaccharide, phorbol myristate and ionomycin (agonists). Gene expression profiles from PBMCs treated with low equi-nicotine units (0.3 µg/mL) of WS-CM and one high dose of STE (100 µg/mL) were similar to those from untreated controls. Cells treated with medium and high doses of WS-CM (1.0 and 3.0 µg/mL) exhibited significantly different gene expression profiles compared to the low WS-CM dose and STE. Pre-treatment with higher doses of WS-CM inhibited the expression of several pro-inflammatory genes (IFNγ, TNFα, and IL-2), while CSF1-R and IL17RA were upregulated. Pre-treatment with high doses of WS-CM abolished agonist-stimulated secretion of IFNγ, TNF and IL-2 proteins. Pathway analyses revealed that higher doses of WS-CM inhibited NF-ĸB signaling, immune cell differentiation and inflammatory responses, and increased apoptotic pathways. Our results show that pre-treatment of PBMCs with higher doses of WS-CM inhibits immune activation and effector cytokine expression and secretion, resulting in a reduced immune response, whereas STE exerted minimal effects.

Urinary Leukotriene E4 and 2,3-Dinor Thromboxane B2 Are Biomarkers of Potential Harm in Short-Term Tobacco Switching Studies.

BACKGROUND: Modified risk tobacco products (MRTP) can reduce harm by decreasing exposure to combustion-related toxicants. In the absence of epidemiologic data, biomarkers of potential harm (BoPH) are useful to evaluate the harm-reducing potential of MRTPs. This study evaluated whether arachidonic acid (AA)-derived metabolites serve as short-term BoPH for predicting harm reduction in tobacco product-switching studies. METHODS: We used 24-hour urine samples from participants in a series of short-term studies in which smokers switched from combustible to noncombustible tobacco products [oral smokeless tobacco products or electronic nicotine delivery system (ENDS)] or abstinence. Pre- and postswitching samples were analyzed by LC/MS-MS for alterations in select AA metabolites, including prostaglandins, isoprostanes, thromboxanes, and leukotrienes. RESULTS: Switching to abstinence, dual use of combustible and noncombustible products, or exclusive use of noncombustible products resulted in reduced 2,3-d-TXB2 levels. Moreover, switching smokers to either abstinence or exclusive use of oral tobacco products resulted in reduced LTE4, but dual use of combustible and oral tobacco products or ENDS did not. A two-biomarker classification model comprising 2,3-d-TXB2 and LTE4 demonstrated the highest performance in distinguishing smokers switched to either abstinence or to ENDS and oral smokeless tobacco products. CONCLUSIONS: Urinary 2,3-d-TXB2 and LTE4 can discriminate between combustible tobacco users and combustible tobacco users switched to either abstinence or noncombustible products for 5 days. IMPACT: 2,3-d-TXB2 and LTE4, which are linked to platelet activation and inflammation, represent BoPH in short-term tobacco product-switching studies. Thus, from a regulatory perspective, 2,3-d-TXB2 and LTE4 may aid in assessing the harm reduction potential of MRTPs.

Global gene expression profiles from PBMCs treated with reference tobacco product preparations.

This Data in Brief article describes global gene expression profiles from human peripheral blood mononuclear cells (PBMCs) that were treated with preparations from reference combustible and non-combustible tobacco products (TPPs). PBMCs isolated from non-smokers were treated with three non-cytotoxic doses of aqueous preparations from 3R4F cigarettes, termed Whole Smoke-Conditioned Medium (WS-CM) and a single dose of 2S3 moist snuff, termed smokeless tobacco extract (STE). PBMCs were treated with the test articles for 3 hours and the extracted total RNA was reverse transcribed and hybridized to HTA 2.0 Genechip® arrays and scanned using an Affymetrix GeneChip® Scanner 3000. CEL files and CHP files were generated using an Affymetrix Expression console. The CEL files were submitted to the NCBI database with GEO accession number GSE110027. The results of the microarray analyses are found in this Data in Brief article. Ingenuity Pathway Analysis (IPA; Qiagen) was used to conduct core analyses of genes that were differentially expressed by high WS-CM or STE based on the Ingenuity Gene knowledge. Expression of several of the differentially expressed genes was confirmed by RT-PCR. Analyses of these data can be found in the article "Distinct gene expression changes in human peripheral blood mononuclear cells treated with different tobacco product preparations" [1].

Combustible Cigarette and Smokeless Tobacco Product Preparations Differentially Regulate Intracellular Calcium Mobilization in HL60 Cells.

Changes in the level of intracellular calcium ([Ca2+]i) are central to leukocyte signaling and immune response. Although evidence suggests that cigarette smoking affects inflammatory response via an increase in intracellular calcium, it remains unclear if the use of smokeless tobacco (e.g., moist snuff) elicits a similar response. In this study, we evaluated the effects of tobacco product preparations (TPPs), including total particulate matter (TPM) from 3R4F reference cigarettes, smokeless tobacco extract (STE) from 2S3 reference moist snuff, and nicotine alone on Ca2+ mobilization in HL60 cells. Treatment with TPM, but not STE or nicotine alone, significantly increased [Ca2+]i in a concentration-dependent manner in HL60 cells. Moreover, TPM-induced [Ca2+]i increase was not related to extracellular Ca2+ and did not require the activation of the IP3 pathway nor involved the transient receptor potential (TRP) channels. Our findings indicate that, in cells having either intact or depleted endoplasmic reticulum (ER) Ca2+ stores, TPM-mediated [Ca2+]i increase involves cytosolic Ca2+ pools other than thapsigargin-sensitive ER Ca2+ stores. These results, for the first time, demonstrate that TPM triggers [Ca2+]i increases, while significantly higher nicotine equivalent doses of STE or nicotine alone, did not affect [Ca2+]i under the experimental conditions. In summary, our study suggests that in contrast with STE or nicotine preparations, TPM activates Ca2+ signaling pathways in HL60 cells. The differential effect of combustible and non-combustible TPPs on Ca2+ mobilization could be a useful in vitro endpoint for tobacco product evaluation.

Distinct gene expression changes in human peripheral blood mononuclear cells treated with different tobacco product preparations.

Cigarette smoking exerts diverse physiological effects including immune suppression. To better characterize the biological effects of different categories of tobacco products, a genome-wide gene expression study was performed. Transcriptomic profiling was performed in PBMCs treated with different equi-nicotine units of aqueous extracts of cigarette smoke (termed Whole Smoke-Conditioned Medium, or WS-CM), or a single dose smokeless tobacco extract (STE) prepared from reference tobacco products. WS-CM induced dose-dependent changes in the expression of several genes. No significant expression differences between low WS-CM and media control were detected. However, transcripts were significantly affected by medium WS-CM (479), high WS-CM (2, 703), and STE (2, 156). The overlap between medium WS-CM and STE, and high WS-CM and STE, was minimal (34 and 65 transcripts, respectively). Hierarchical clustering revealed that gene expression profiles for STE and medium WS-CM co-clustered, while those affected by the high dose of WS-CM clustered distinctly. Functional analysis revealed that WS-CM, but not STE, uniquely affected genes involved in immune cell development and inflammatory response. Cascades of upstream regulators (e.g., TNF, IL1ß, NFƙB) were identified for the observed gene expression changes and generally suppressed by WS-CM, but not by STE. Collectively, these findings demonstrate that combustible and non-combustible tobacco products elicit distinct biological effects, which could explain the observed chronic immune suppression in smokers.

Suppression of Neutrophil Antimicrobial Functions by Total Particulate Matter From Cigarette Smoke.

Chronic cigarette smoking is widely known to alter immune functions and compromise host defense against microbial infection. Neutrophils play an essential role in the immune defense against microbial pathogens and also participate in the development of the inflammatory responses. However, there is limited information about the effects of cigarette smoking on neutrophil response. In this study, cultured bone marrow neutrophils were exposed to total particulate matter (TPM) from cigarette smoke. We found that TPM not only reduced LPS-induced TNFα production, but also suppressed neutrophil bactericidal activity. We also observed that TPM priming reduced the expression of NADPH oxidase component gp91 and iNOS, molecules important for bacterial killing. Mechanistically, we documented that TPM-primed neutrophils have reduced STAT1 activation following subsequent LPS challenge. STAT1 is a key transcription factor responsible for the expression of inflammatory genes as well as gp91 and iNOS. Collectively, reduced STAT1 activation and reduced NADPH oxidase/iNOS may potentially explain the compromised anti-microbial function of TPM-programmed neutrophils. Taken together, our findings reveal that the key innate immune neutrophil is subject to reprogramming by smoking to adopt an immune-suppressed state, potentially responsible for chronic smoking-mediated immunosuppression.

Outcomes of developmental exposure to total particulate matter from cigarette smoke in zebrafish (Danio rerio).

The effects of prenatal exposure to cigarette smoke remain a subject of major interest, especially as it relates to neural development and adverse behavioral outcomes. Several studies have investigated the developmental toxicity of cigarette smoke components in a zebrafish model, showing that developmental exposure to total particulate matter (TPM; particulate phase of cigarette smoke) leads to adverse physiological aberrations and locomotor hyperactivity. Thus, the current study examines whether developmental TPM exposure of zebrafish embryos/larvae (F0) leads to physiological and behavioral alterations, and whether adverse effects are observed in adult fish and the next generation (F1; i.e. F0 offspring). We also examine whether behavioral effects are associated with changes in neural development, stress response, neurotransmitters, and bioenergetics. We demonstrate that TPM exposure during F0 development increased the incidence of deformities in F0 larvae, but F1 larvae did not exhibit any deformities. TPM exposure also resulted in swimming hyperactivity in F0 larvae and several behavioral changes were noted in F0 fish when they grew into adulthood. These behavioral changes were generally not associated with changes in markers of neural development in larvae, stress response in F0 adults, and concentration of neurotransmitters (acetylcholine, dopamine, and serotonin) in F0 adult brain. There were also no changes in F0 or F1 embryonic oxygen consumption rate (OCR; marker of bioenergetics and mitochondrial health); however, the OCR in the brain of F0 males was reduced with TPM. We conclude that developmental exposure to TPM affects larval physiology and induces hyperactive swimming behavior, but these effects do not persist in F1 larvae. Moreover, developmental TPM exposure leads to long-lasting sex-specific behavioral outcomes in the F0 adult fish.

Global methylation profiles in buccal cells of long-term smokers and moist snuff consumers.

PURPOSE: Alternations in gene methylation and other epigenetic changes regulate normal development as well as drive disease progression. The aim of this study is to investigate global methylation changes in the buccal cells of smokers and smokeless tobacco users. MATERIALS AND METHODS: Generally healthy adult male subjects were recruited into smoker (SMK), moist snuff consumer (MSC) and non-tobacco consumer (NTC) cohorts (40 subjects/cohort) (ClinicalTrials.gov Identifier: NCT01923402). Global methylation profiling was performed on Illumina 450 K methylation arrays using buccal cell DNAs. RESULTS: The SMK cohort exhibited larger qualitative and quantitative changes relative to MSC. Approximately half of the differentially methylated 1252 gene loci were grouped as combustible tobacco-related (CTR) signatures and a third of the changes, tobacco-related (TR) signatures, were associated with smoking. Very few (41) differentially methylated gene loci were exclusively associated with moist snuff use and were designated as moist snuff-related (MSR) signature. Pathway enrichment analyses revealed that developmental and immune response pathways, among others, were impacted due to tobacco use. CONCLUSIONS: Chronic cigarette smoking causes hyper- and hypo-methylation of genes that could contribute to smoking-related diseases. These results help place combustible and non-combustible tobacco products along a risk continuum and provide additional insights into the effects of tobacco consumption.

Total particulate matter from cigarette smoke disrupts vascular development in zebrafish brain (Danio rerio).

Several studies have demonstrated zebrafish as a useful high-throughput in vivo model to study the effects of cigarette smoke on early development. It has been shown previously that exposure of zebrafish to cigarette smoke total particulate matter (TPM) leads to several adverse physiological aberrations, including heart deformities and improper angiogenesis. Consequently, this study investigated the effects of TPM on cardiovascular development in zebrafish that were exposed to increasing concentrations of TPM based upon nicotine content from 6h post fertilization (hpf) up to 72hpf. We show that TPM exposure in wild-type embryos led to a dose-dependent increase in fluorescence, especially in the yolk and head regions, suggesting bioaccumulation of cyclic compounds in TPM, such as polycyclic aromatic hydrocarbons (PAHs). Similarly, the incidence of cranial hemorrhage, pericardial edema, and string heart was increased with TPM exposure in a dose-dependent manner. Additionally, TPM exposure in transgenic (Flk1:eGFP) zebrafish showed a decrease in vascular abundance in the brain, but the transcript abundance of key angiogenic genes Tie-2, Angpt1, Notch3, and Flk1 remained largely unchanged and that of Vegf actually increased with TPM. The study also investigated aspects of a proposed crosstalk between the activation of the aryl hydrocarbon receptor (AhR) pathway and subsequent inhibition of the Wnt signaling pathway, resulting in cardiac malformations. In an effort to reduce the occurrence of cardiovascular malformations, embryos/larvae were co-treated with CHIR99021 (CHIR), which should promote Wnt signaling. However, co-treatment with CHIR did not significantly affect the TPM-induced cardiovascular toxicity. Overall, results from this study demonstrate that exposure to TPM leads to several cardiovascular deformities and disrupted vascular development in the brain, and that these effects are associated with downregulation of Wnt signaling.

Inflammatory profile analysis reveals differences in cytokine expression between smokers, moist snuff users, and dual users compared to non-tobacco consumers.

OBJECTIVE AND DESIGN: The aim of this study is to investigate the inflammatory alterations due to the use of smokeless tobacco and dual use of smokeless tobacco and cigarettes, relative to smoking. SUBJECTS: Plasma and saliva samples were collected from healthy smokers (SMK-100 subjects), moist snuff users (MSC-89 subjects), the dual users (DUSMK-49 subjects), and non-tobacco consumers (NTC-99 subjects) from two cross-sectional studies. METHODS: Luminex Human InflammationMAP® 1.0 panel, a multiplex immunoassay. RESULTS: SMK and DUSMK exhibited larger number of alterations in the expression of inflammatory analytes compared to NTC. Eight analytes were significantly elevated (p ≤ .05) within plasma samples of SMK compared to NTC, while one 1 analyte was elevated between the MSC and NTC groups. DUSMK exhibited different levels of 11 analytes, relative to NTC. MSC displayed fewer alterations in inflammatory protein expression compared to smoker groups, and the inflammatory profile of MSC resembles NTC. Five analytes (ICAM-1, VEGF, MMP-9, ferritin and fibrinogen) emerged as potential biomarkers distinguishing tobacco consumers (p < .02). CONCLUSIONS: We identified a set of five proteins as potential biomarkers that can inform of inflammation status due to tobacco usage. Our findings contribute a better understanding of how the use of different tobacco products contributes to inflammation.

Role of Oxidative Stress in the Suppression of Immune Responses in Peripheral Blood Mononuclear Cells Exposed to Combustible Tobacco Product Preparation.

Cigarette smoking is a major risk factor for several human diseases. Chronic inflammation, resulting from increased oxidative stress, has been suggested as a mechanism that contributes to the increased susceptibility of smokers to cancer and microbial infections. We have previously shown that whole-smoke conditioned medium (WS-CM) and total particulate matter (TPM) prepared from Kentucky 3R4F reference cigarettes [collectively called as combustible tobacco product preparations (TPPs)] potently suppressed agonist-stimulated cytokine secretion and target cell killing in peripheral blood mononuclear cells (PBMCs). Here we have investigated the role of oxidative stress from TPPs, which alters inflammatory responses in vitro. Particularly, we investigated the mechanisms of WS-CM-induced suppression of select cytokine secretions in Toll-like receptor (TLR) agonist-stimulated cells and target cell killing by effector cells in PBMCs. Pretreatment with N-acetyl cysteine (NAC), a precursor of reduced glutathione and an established anti-oxidant, protected against DNA damage and cytotoxicity caused by exposure to WS-CM. Similarly, secretion of tumor necrosis factor (TNF), interleukin (IL)-6, and IL-8 in response to TLR-4 stimulation was restored by pretreatment with NAC. Target cell killing, a functional measure of cytolytic cells in PBMCs, is suppressed by WS-CM. Pretreatment with NAC restored the target cell killing in WS-CM treated PBMCs. This was accompanied by higher perforin levels in the effector cell populations. Collectively, these data suggest that reducing oxidative stress caused by cigarette smoke components restores select immune responses in this ex vivo model.

AKR1C1 as a Biomarker for Differentiating the Biological Effects of Combustible from Non-Combustible Tobacco Products.

Smoking has been established as a major risk factor for developing oral squamous cell carcinoma (OSCC), but less attention has been paid to the effects of smokeless tobacco products. Our objective is to identify potential biomarkers to distinguish the biological effects of combustible tobacco products from those of non-combustible ones using oral cell lines. Normal human gingival epithelial cells (HGEC), non-metastatic (101A) and metastatic (101B) OSCC cell lines were exposed to different tobacco product preparations (TPPs) including cigarette smoke total particulate matter (TPM), whole-smoke conditioned media (WS-CM), smokeless tobacco extract in complete artificial saliva (STE), or nicotine (NIC) alone. We performed microarray-based gene expression profiling and found 3456 probe sets from 101A, 1432 probe sets from 101B, and 2717 probe sets from HGEC to be differentially expressed. Gene Set Enrichment Analysis (GSEA) revealed xenobiotic metabolism and steroid biosynthesis were the top two pathways that were upregulated by combustible but not by non-combustible TPPs. Notably, aldo-keto reductase genes, AKR1C1 and AKR1C2, were the core genes in the top enriched pathways and were statistically upregulated more than eight-fold by combustible TPPs. Quantitative real time polymerase chain reaction (qRT-PCR) results statistically support AKR1C1 as a potential biomarker for differentiating the biological effects of combustible from non-combustible tobacco products.

Gene expression profiles associated with cigarette smoking and moist snuff consumption.

BACKGROUND: Among the different tobacco products that are available on the US market, cigarette smoking is shown to be the most harmful and the effects of cigarette smoking have been well studied. US epidemiological studies indicate that non-combustible tobacco products are less harmful than smoking and yet very limited biological and mechanistic information is available on the effects of these alternative tobacco products. For the first time, we characterized gene expression profiling in PBMCs from moist snuff consumers (MSC), compared with that from consumers of cigarettes (SMK) and non-tobacco consumers (NTC). RESULTS: Microarray analysis identified 100 differentially expressed genes (DEGs) between the SMK and NTC groups and 46 DEGs between SMK and MSC groups. However, we found no significant differences in gene expression between MSC and NTC. Both hierarchical clustering and principle component analysis revealed that MSC and NTC expression profiles were more similar than to SMK. Random forest classification identified a subset of DEGs which predicted SMK from either NTC or MSC with high accuracy (AUC 0.98). CONCLUSIONS: PMBC gene expression profiles of NTC and MSC are similar to each other, while SMK exhibit distinct profiles with alterations in immune related pathways. In addition to discovering several biomarkers, these studies support further understanding of the biological effects of different tobacco products. TRIAL REGISTRATION: ClinicalTrials.gov. Identifier: NCT01923402 . Date of Registration: August 14, 2013. Study was retrospectively registered.