Publications by authors named "Karsta Luettich"

30 Publications

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A 6-month inhalation toxicology study in Apoe mice demonstrates substantially lower effects of e-vapor aerosol compared with cigarette smoke in the respiratory tract.

Arch Toxicol 2021 05 7;95(5):1805-1829. Epub 2021 May 7.

PMI R&D, Philip Morris Products S.A, Quai Jeanrenaud 5, 2000, Neuchâtel, Switzerland.

Cigarette smoking is the major cause of chronic obstructive pulmonary disease. Considerable attention has been paid to the reduced harm potential of nicotine-containing inhalable products such as electronic cigarettes (e-cigarettes). We investigated the effects of mainstream cigarette smoke (CS) and e-vapor aerosols (containing nicotine and flavor) generated by a capillary aerosol generator on emphysematous changes, lung function, and molecular alterations in the respiratory system of female Apoe mice. Mice were exposed daily (3 h/day, 5 days/week) for 6 months to aerosols from three different e-vapor formulations-(1) carrier (propylene glycol and vegetable glycerol), (2) base (carrier and nicotine), or (3) test (base and flavor)-or to CS from 3R4F reference cigarettes. The CS and base/test aerosol concentrations were matched at 35 µg nicotine/L. CS exposure, but not e-vapor exposure, led to impairment of lung function (pressure-volume loop area, A and K parameters, quasi-static elastance and compliance) and caused marked lung inflammation and emphysematous changes, which were confirmed histopathologically and morphometrically. CS exposure caused lung transcriptome (activation of oxidative stress and inflammatory responses), lipidome, and proteome dysregulation and changes in DNA methylation; in contrast, these effects were substantially reduced in response to the e-vapor aerosol exposure. Compared with sham, aerosol exposure (carrier, base, and test) caused a slight impact on lung inflammation and epithelia irritation. Our results demonstrated that, in comparison with CS, e-vapor aerosols induced substantially lower biological and pathological changes in the respiratory tract associated with chronic inflammation and emphysema.
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http://dx.doi.org/10.1007/s00204-021-03020-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113187PMC
May 2021

Development of an Advanced Multicellular Intestinal Model for Assessing Immunomodulatory Properties of Anti-Inflammatory Compounds.

Front Pharmacol 2021 16;12:639716. Epub 2021 Apr 16.

PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland.

Intestinal inflammation is the collective term for immune system-mediated diseases of unknown, multifactorial etiology, with often complex interactions between genetic and environmental factors. To mechanistically investigate the effect of treatment with compounds possessing immunomodulating properties in the context of intestinal inflammation, we developed an immunocompetent triculture intestinal model consisting of a differentiated intestinal epithelial layer (Caco-2/HT29-MTX) and immunocompetent cells (differentiated THP-1). The triculture mimicked a healthy intestine with stable barrier integrity. Lipopolysaccharide treatment triggered a controlled and reversible inflammatory state, resulting in significant impairment of barrier integrity and release of pro-inflammatory cytokines and chemokines, which are known hallmarks of intestinal inflammation. Treatment with known anti-inflammatory reference compounds (TPCA-1 and budenoside) prevented the induction of an inflammatory state; the decreasing triculture responses to this treatment measured by cytokine release, transepithelial electric resistance (TEER), and epithelial layer permeability proved the suitability of the intestinal model for anti-inflammatory drug screening. Finally, selected tobacco alkaloids (nicotine and anatabine (/ and forms)) were tested in the triculture for their potential anti-inflammatory properties. Indeed, naturally occurring alkaloids, such as tobacco-derived alkaloids, have shown substantial anti-inflammatory effects in several and models of inflammation, gaining increasing interest. Similar to the anti-inflammatory reference compounds, one of the tobacco alkaloids under investigation partially prevented the decrease in the TEER and increase in permeability and reduced the release of pro-inflammatory cytokines and chemokines. Taken together, these data confirm that our model is suitable for screening potential anti-inflammatory compounds in the context of intestinal inflammation.
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http://dx.doi.org/10.3389/fphar.2021.639716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085553PMC
April 2021

Respiratory Effects of Exposure to Aerosol From the Candidate Modified-Risk Tobacco Product THS 2.2 in an 18-Month Systems Toxicology Study With A/J Mice.

Toxicol Sci 2020 11;178(1):138-158

PMI R&D, Philip Morris Products S.A, CH-2000 Neuchâtel, Switzerland.

Smoking cessation is the most effective measure for reducing the risk of smoking-related diseases. However, switching to less harmful products (modified-risk tobacco products [MRTP]) can be an alternative to help reduce the risk for adult smokers who would otherwise continue to smoke. In an 18-month chronic carcinogenicity/toxicity study in A/J mice (OECD Test Guideline 453), we assessed the aerosol of Tobacco Heating System 2.2 (THS 2.2), a candidate MRTP based on the heat-not-burn principle, compared with 3R4F cigarette smoke (CS). To capture toxicity- and disease-relevant mechanisms, we complemented standard toxicology endpoints with in-depth systems toxicology analyses. In this part of our publication series, we report on integrative assessment of the apical and molecular exposure effects on the respiratory tract (nose, larynx, and lungs). Across the respiratory tract, we found changes in inflammatory response following 3R4F CS exposure (eg, antimicrobial peptide response in the nose), with both shared and distinct oxidative and xenobiotic responses. Compared with 3R4F CS, THS 2.2 aerosol exerted far fewer effects on respiratory tract histology, including adaptive tissue changes in nasal and laryngeal epithelium and inflammation and emphysematous changes in the lungs. Integrative analysis of molecular changes confirmed the substantially lower impact of THS 2.2 aerosol than 3R4F CS on toxicologically and disease-relevant molecular processes such as inflammation, oxidative stress responses, and xenobiotic metabolism. In summary, this work exemplifies how apical and molecular endpoints can be combined effectively for toxicology assessment and further supports findings on the reduced respiratory health risks of THS 2.2 aerosol.
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http://dx.doi.org/10.1093/toxsci/kfaa132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657339PMC
November 2020

Reduced Chronic Toxicity and Carcinogenicity in A/J Mice in Response to Life-Time Exposure to Aerosol From a Heated Tobacco Product Compared With Cigarette Smoke.

Toxicol Sci 2020 11;178(1):44-70

PMI R&D, Philip Morris International Research Laboratories Pte. Ltd, Science Park II, Singapore 117406, Singapore.

We conducted an inhalation study, in accordance with Organisation for Economic Co-operation and Development Test Guideline 453, exposing A/J mice to tobacco heating system (THS) 2.2 aerosol or 3R4F reference cigarette smoke (CS) for up to 18 months to evaluate chronic toxicity and carcinogenicity. All exposed mice showed lower thymus and spleen weight, blood lymphocyte counts, and serum lipid concentrations than sham mice, most likely because of stress and/or nicotine effects. Unlike THS 2.2 aerosol-exposed mice, CS-exposed mice showed increased heart weight, changes in red blood cell profiles and serum liver function parameters. Similarly, increased pulmonary inflammation, altered lung function, and emphysematous changes were observed only in CS-exposed mice. Histopathological changes in other respiratory tract organs were significantly lower in the THS 2.2 aerosol-exposed groups than in the CS-exposed group. Chronic exposure to THS 2.2 aerosol also did not increase the incidence or multiplicity of bronchioloalveolar adenomas or carcinomas relative to sham, whereas CS exposure did. Male THS 2.2 aerosol-exposed mice had a lower survival rate than sham mice, related to an increased incidence of urogenital issues that appears to be related to congenital factors rather than test item exposure. The lower impact of THS 2.2 aerosol exposure on tumor development and chronic toxicity is consistent with the significantly reduced levels of harmful and potentially harmful constituents in THS 2.2 aerosol relative to CS. The totality of the evidence from this study further supports the risk reduction potential of THS 2.2 for lung diseases in comparison with cigarettes.
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http://dx.doi.org/10.1093/toxsci/kfaa131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657344PMC
November 2020

Human multi-organ chip co-culture of bronchial lung culture and liver spheroids for substance exposure studies.

Sci Rep 2020 05 12;10(1):7865. Epub 2020 May 12.

PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000, Neuchâtel, Switzerland.

Extrapolation of cell culture-based test results to in vivo effects is limited, as cell cultures fail to emulate organ complexity and multi-tissue crosstalk. Biology-inspired microphysiological systems provide preclinical insights into absorption, distribution, metabolism, excretion, and toxicity of substances in vitro by using human three-dimensional organotypic cultures. We co-cultured a human lung equivalent from the commercially available bronchial MucilAir culture and human liver spheroids from HepaRG cells to assess the potential toxicity of inhaled substances under conditions that permit organ crosstalk. We designed a new HUMIMIC Chip with optimized medium supply and oxygenation of the organ cultures and cultivated them on-chip for 14 days in separate culture compartments of a closed circulatory perfusion system, demonstrating the viability and homeostasis of the tissue cultures. A single-dose treatment of the hepatotoxic and carcinogenic aflatoxin B impaired functionality in bronchial MucilAir tissues in monoculture but showed a protective effect when the tissues were co-cultured with liver spheroids, indicating that crosstalk can be achieved in this new human lung-liver co-culture. The setup described here may be used to determine the effects of exposure to inhaled substances on a systemic level.
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http://dx.doi.org/10.1038/s41598-020-64219-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217973PMC
May 2020

Mitochondrial Network and Biogenesis in Response to Short and Long-Term Exposure of Human BEAS-2B Cells to Aerosol Extracts from the Tobacco Heating System 2.2.

Cell Physiol Biochem 2020 Mar;54(2):230-251

Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland,

Background/aims: Adverse effects of cigarette smoke on health are widely known. Heating rather than combusting tobacco is one of strategies to reduce the formation of toxicants. The sensitive nature of mitochondrial dynamics makes the mitochondria an early indicator of cellular stress. For this reason, we studied the morphology and dynamics of the mitochondrial network in human bronchial epithelial cells (BEAS-2B) exposed to total particulate matter (TPM) generated from 3R4F reference cigarette smoke and from aerosol from a new candidate modified risk tobacco product, the Tobacco Heating System (THS 2.2).

Methods: Cells were subjected to short (1 week) and chronic (12 weeks) exposure to a low (7.5 µg/mL) concentration of 3R4F TPM and low (7.5 µg/mL), medium (37.5 µg/mL), and high (150 µg/mL) concentrations of TPM from THS 2.2. Confocal microscopy was applied to assess cellular and mitochondrial morphology. Cytosolic Ca levels, mitochondrial membrane potential and mitochondrial mass were measured with appropriate fluorescent probes on laser scanning cytometer. The levels of proteins regulating mitochondrial dynamics and biogenesis were determined by Western blot.

Results: In BEAS-2B cells exposed for one week to the low concentration of 3R4F TPM and the high concentration of THS 2.2 TPM we observed clear changes in cell morphology, mitochondrial network fragmentation, altered levels of mitochondrial fusion and fission proteins and decreased biogenesis markers. Also cellular proliferation was slowed down. Upon chronic exposure (12 weeks) many parameters were affected in the opposite way comparing to short exposure. We observed strong increase of NRF2 protein level, reorganization of mitochondrial network and activation of the mitochondrial biogenesis process.

Conclusion: Comparison of the effects of TPMs from 3R4F and from THS 2.2 revealed, that similar extent of alterations in mitochondrial dynamics and biogenesis is observed at 7.5 µg/mL of 3R4F TPM and 150 µg/mL of THS 2.2 TPM. 7 days exposure to the investigated components of cigarette smoke evoke mitochondrial stress, while upon chronic, 12 weeks exposure the hallmarks of cellular adaptation to the stressor were visible. The results also suggest that mitochondrial stress signaling is involved in the process of cellular adaptation under conditions of chronic stress caused by 3R4F and high concentration of THS 2.2.
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http://dx.doi.org/10.33594/000000216DOI Listing
March 2020

A 6-month systems toxicology inhalation study in ApoE mice demonstrates reduced cardiovascular effects of E-vapor aerosols compared with cigarette smoke.

Am J Physiol Heart Circ Physiol 2020 03 24;318(3):H604-H631. Epub 2020 Jan 24.

Philip Morris International Research and Development, Philip Morris Products, Neuchâtel, Switzerland.

Smoking cigarettes is harmful to the cardiovascular system. Considerable attention has been paid to the reduced harm potential of alternative nicotine-containing inhalable products such as e-cigarettes. We investigated the effects of E-vapor aerosols or cigarette smoke (CS) on atherosclerosis progression, cardiovascular function, and molecular changes in the heart and aorta of female apolipoprotein E-deficient (ApoE) mice. The mice were exposed to aerosols from three different E-vapor formulations: ) carrier (propylene glycol and vegetable glycerol), ) base (carrier and nicotine), or ) test (base and flavor) or to CS from 3R4F reference cigarettes for up to 6 mo. Concentrations of CS and base or test aerosols were matched at 35 µg nicotine/L. Exposure to CS, compared with sham-exposed fresh air controls, accelerated atherosclerotic plaque formation, whereas no such effect was seen for any of the three E-vapor aerosols. Molecular changes indicated disease mechanisms related to oxidative stress and inflammation in general, plus changes in calcium regulation, and altered cytoskeletal organization and microtubule dynamics in the left ventricle. While ejection fraction, fractional shortening, cardiac output, and isovolumic contraction time remained unchanged following E-vapor aerosols exposure, the nicotine-containing base and test aerosols caused an increase in isovolumic relaxation time similar to CS. A nicotine-related increase in pulse wave velocity and arterial stiffness was also observed, but it was significantly lower for base and test aerosols than for CS. These results demonstrate that in comparison with CS, E-vapor aerosols induce substantially lower biological responses associated with smoking-related cardiovascular diseases. Analysis of key urinary oxidative stress markers and proinflammatory cytokines showed an absence of oxidative stress and inflammation in the animals exposed to E-vapor aerosols. Conversely, animals exposed to conventional cigarette smoke had high urinary levels of these markers. When compared with conventional cigarette smoke, E-vapor aerosols induced smaller atherosclerotic plaque surface area and volume. Systolic and diastolic cardiac function, as well as endothelial function, were further significantly less affected by electronic cigarette aerosols than conventional cigarette smoke. Molecular analysis demonstrated that E-vapor aerosols induce significantly smaller transcriptomic dysregulation in the heart and aorta compared with conventional cigarette smoke.
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http://dx.doi.org/10.1152/ajpheart.00613.2019DOI Listing
March 2020

In Vitro High-Content Imaging-Based Phenotypic Analysis of Bronchial 3D Organotypic Air-Liquid Interface Cultures.

SLAS Technol 2020 Jun 23;25(3):247-252. Epub 2020 Jan 23.

PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland.

High-content imaging (HCI) is a powerful method for quantifying biological effects in vitro. Historically, HCI has been applied to adherent cells growing in monolayers. With the advent of confocal versions of HCI devices, researchers now have the option of performing analyses on 3D cell cultures. However, some obstacles remain in integrating the third dimension, such as limited light penetration and less sophisticated image analysis. Here, we report the development of an HCI technique for imaging human bronchial 3D organotypic air-liquid interface (ALI) cultures (hBR-ALI). In this method, we monitored differentiation status through HCI evaluation markers representative of ciliated epithelial cells and goblet cells (Muc5AC [mucin 5AC]). As a second use case for demonstrating the utility of this technique, we induced goblet cell hyperplasia in hBR-ALI by using interleukin (IL)-13. Our results demonstrate the utility of the HCI technique for imaging hBR-ALI grown on Transwell inserts. This technique may be expanded to other cell culture systems, such as skin epithelia and 3D intestinal systems.
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http://dx.doi.org/10.1177/2472630319895473DOI Listing
June 2020

Mitochondria as a possible target for nicotine action.

J Bioenerg Biomembr 2019 08 13;51(4):259-276. Epub 2019 Jun 13.

Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093, Warsaw, Poland.

Mitochondria are multifunctional and dynamic organelles deeply integrated into cellular physiology and metabolism. Disturbances in mitochondrial function are involved in several disorders such as neurodegeneration, cardiovascular diseases, metabolic diseases, and also in the aging process. Nicotine is a natural alkaloid present in the tobacco plant which has been well studied as a constituent of cigarette smoke. It has also been reported to influence mitochondrial function both in vitro and in vivo. This review presents a comprehensive overview of the present knowledge of nicotine action on mitochondrial function. Observed effects of nicotine exposure on the mitochondrial respiratory chain, oxidative stress, calcium homeostasis, mitochondrial dynamics, biogenesis, and mitophagy are discussed, considering the context of the experimental design. The potential action of nicotine on cellular adaptation and cell survival is also examined through its interaction with mitochondria. Although a large number of studies have demonstrated the impact of nicotine on various mitochondrial activities, elucidating its mechanism of action requires further investigation.
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http://dx.doi.org/10.1007/s10863-019-09800-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679833PMC
August 2019

Construction of a Suite of Computable Biological Network Models Focused on Mucociliary Clearance in the Respiratory Tract.

Front Genet 2019 15;10:87. Epub 2019 Feb 15.

PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland.

Mucociliary clearance (MCC), considered as a collaboration of mucus secreted from goblet cells, the airway surface liquid layer, and the beating of cilia of ciliated cells, is the airways' defense system against airborne contaminants. Because the process is well described at the molecular level, we gathered the available information into a suite of comprehensive causal biological network (CBN) models. The suite consists of three independent models that represent (1) cilium assembly, (2) ciliary beating, and (3) goblet cell hyperplasia/metaplasia and that were built in the Biological Expression Language, which is both human-readable and computable. The network analysis of highly connected nodes and pathways demonstrated that the relevant biology was captured in the MCC models. We also show the scoring of transcriptomic data onto these network models and demonstrate that the models capture the perturbation in each dataset accurately. This work is a continuation of our approach to use computational biological network models and mathematical algorithms that allow for the interpretation of high-throughput molecular datasets in the context of known biology. The MCC network model suite can be a valuable tool in personalized medicine to further understand heterogeneity and individual drug responses in complex respiratory diseases.
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http://dx.doi.org/10.3389/fgene.2019.00087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384416PMC
February 2019

How complex should an in vitro model be? Evaluation of complex 3D alveolar model with transcriptomic data and computational biological network models.

ALTEX 2019 12;36(3):388-402. Epub 2019 Feb 12.

PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland.

To more accurately model inhalation toxicity in vitro, we developed a tetra-culture system that combines lung alveolar epithelial cells, endothelial cells, macrophages, and mast cells in a three-dimensional orientation. We characterized the influence of the added complexity using network perturbation analysis and gene expression data. This will allow us to gain insight into the steady-state profile of the assembled, complete three-dimensional model using all four cell types and of simpler models of one, two, or three cell types. Gene expression data were analyzed using cause-and-effect biological network models, together with a quantitative network-scoring algorithm, to determine the biological impact of co-culturing the various cell types. In the assembled tetra-culture, macrophages appeared to be the largest contributors to overall network perturbations, promoting high basal levels of oxidative stress and inflammation. This finding led to further optimization of the model using rested macrophages; the addition of rested macrophages decreased the basal inflammatory and cell stress status of the co-culture. Finally, we compared transcriptional profiles from publicly available datasets of conventional in vitro models representative of the airways and of healthy human lung tissues to assess similarities between our model and other in vitro models and the human lung. On the transcriptional level, we found an increasing correlation between airway models and normal human lung tissue, particularly as cell types became more physiologically relevant and the complexity of the system increased. This indicates that the combination of multiple lung-relevant cell types in vitro does indeed increase similarity to the physiological counterpart.
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http://dx.doi.org/10.14573/altex.1811221DOI Listing
January 2020

A lung/liver-on-a-chip platform for acute and chronic toxicity studies.

Lab Chip 2018 12;18(24):3814-3829

PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchâtel, Switzerland.

The merging of three-dimensional in vitro models with multi-organ-on-a-chip (MOC) technology has taken in vitro assessment of chemicals to an unprecedented level. By connecting multiple organotypic models, MOC allows for the crosstalk between different organs to be studied to evaluate a compound's safety and efficacy better than with single cultures. The technology could also improve the toxicological assessment of aerosols that have been implicated in the development of chronic obstructive pulmonary disease, asthma, or lung cancer. Here we report the development of a lung/liver-on-a-chip, connecting in a single circuit, normal human bronchial epithelial (NHBE) cells cultured at the air-liquid interface (ALI), and HepaRG™ liver spheroids. Maintenance of the individual tissues in the chip increased NHBE ALI tissue transepithelial electrical resistance and decreased HepaRG™ spheroid adenosine triphosphate content as well as cytochrome P450 (CYP) 1A1/1B1 inducibility. CYP inducibility was partly restored when HepaRG™ spheroids were cocultured with NHBE ALI tissues. Both tissues remained viable and functional for 28 days when cocultured in the chip. The capacity of the HepaRG™ spheroids to metabolize compounds present in the medium and to modulate their toxicity was proven using aflatoxin B1 (AFB1). AFB1 toxicity in NHBE ALI tissues decreased when HepaRG™ spheroids were present in the same chip circuit, proving that the HepaRG™-mediated detoxification is protecting/decreasing from AFB1-mediated cytotoxicity. The lung/liver-on-a-chip platform presented here offers new opportunities to study the toxicity of inhaled aerosols or to demonstrate the safety and efficacy of new drug candidates targeting the human lung.
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http://dx.doi.org/10.1039/c8lc01029cDOI Listing
December 2018

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity.

Toxicol In Vitro 2018 Oct 20;52:131-145. Epub 2018 Jun 20.

Procter & Gamble Co, 11530 Reed Hartman Highway, Cincinnati, OH 45241, United States.

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances.
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http://dx.doi.org/10.1016/j.tiv.2018.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760245PMC
October 2018

Physiological and biological characterization of smokers with and without COPD.

F1000Res 2017 13;6:877. Epub 2017 Jun 13.

Philip Morris Products SA, Philip Morris International R&D, Quai Jeanrenaud 5, 2000 Neuchâtel, Switzerland.

Chronic obstructive pulmonary disease (COPD) is a common inflammatory airway disease predominantly associated with cigarette smoking, and its incidence is increasing worldwide. According to the Global Initiative for Obstructive Lung Disease (GOLD) guidelines, spirometry is used to diagnose the disease. However, owing to its complexity, spirometry alone may not account for the multitude of COPD phenotypes or the early, asymptomatic lung damage seen in younger smokers. In addition, suitable biomarkers enabling early diagnosis, guiding treatment and estimating prognosis are still scarce, although large scale 'omics analyses have added to the spectrum of potential biomarkers that could be used for these purposes. The aim of the current study was to comprehensively profile patients with mild-to-moderate COPD and compare the profiles to i) a group of currently smoking asymptomatic subjects, ii) a group of healthy former smokers, and iii) a group of healthy subjects that had never smoked. The assessment was conducted at the molecular level using proteomics, transcriptomics, and lipidomics and complemented by a series of measurements of traditional and emerging indicators of lung health (ClinicalTrials.gov identifier: NCT01780298). In this data note, we provide a comprehensive description of the study population's physiological characteristics including full lung function, lung appearance on chest computed tomography, impulse oscillometry, and exercise tolerance and quality of life (QoL) measures.
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http://dx.doi.org/10.12688/f1000research.11698.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5843826PMC
June 2017

The biological effects of long-term exposure of human bronchial epithelial cells to total particulate matter from a candidate modified-risk tobacco product.

Toxicol In Vitro 2018 Aug 7;50:95-108. Epub 2018 Mar 7.

PMI R&D, Philip Morris Products S.A. (Part of Philip Morris International group of companies), Quai Jeanrenaud 5, CH-2000 Neuchâtel, Switzerland.

Cigarette smoking is the leading cause of preventable lung cancer (LC). Reduction of harmful constituents by heating rather than combusting tobacco may have the potential to reduce the risk of LC. We evaluated functional and molecular changes in human bronchial epithelial BEAS-2B cells following a 12-week exposure to total particulate matter (TPM) from the aerosol of a candidate modified-risk tobacco product (cMRTP) in comparison with those following exposure to TPM from the 3R4F reference cigarette. Endpoints linked to lung carcinogenesis were assessed. Four-week 3R4F TPM exposure resulted in crisis and epithelial to mesenchymal transition (EMT) accompanied by decreased barrier function and disrupted cell-to-cell contacts. By week eight, cells regained E-cadherin expression, suggesting that EMT was reversible. Increased levels of inflammatory mediators were noted in cells treated to 3R4F TPM but not in cells treated to the same or a five-fold higher concentration of cMRTP TPM. A 20-fold higher concentration of cMRTP TPM increased oxidative stress and DNA damage and caused reversible EMT. Anchorage-independent growth was observed in cells treated to 3R4F or a high concentration of cMRTP TPM. 3R4F TPM-derived clones were invasive, while cMRTP TPM-derived clones were not. Long-term exposure to TPM from the cMRTP had a lower biological impact on BEAS-2B cells compared with that of exposure to TPM from 3R4F.
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http://dx.doi.org/10.1016/j.tiv.2018.02.019DOI Listing
August 2018

Assessment of mitochondrial function following short- and long-term exposure of human bronchial epithelial cells to total particulate matter from a candidate modified-risk tobacco product and reference cigarettes.

Food Chem Toxicol 2018 May 13;115:1-12. Epub 2018 Feb 13.

Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warsaw, Poland. Electronic address:

Mitochondrial dysfunction caused by cigarette smoke is involved in the oxidative stress-induced pathology of airway diseases. Reducing the levels of harmful and potentially harmful constituents by heating rather than combusting tobacco may reduce mitochondrial changes that contribute to oxidative stress and cell damage. We evaluated mitochondrial function and oxidative stress in human bronchial epithelial cells (BEAS 2B) following 1- and 12-week exposures to total particulate matter (TPM) from the aerosol of a candidate modified-risk tobacco product, the Tobacco Heating System 2.2 (THS2.2), in comparison with TPM from the 3R4F reference cigarette. After 1-week exposure, 3R4F TPM had a strong inhibitory effect on mitochondrial basal and maximal oxygen consumption rates compared to TPM from THS2.2. Alterations in oxidative phosphorylation were accompanied by increased mitochondrial superoxide levels and increased levels of oxidatively damaged proteins in cells exposed to 7.5 μg/mL of 3R4F TPM or 150 μg/mL of THS2.2 TPM, while cytosolic levels of reactive oxygen species were not affected. In contrast, the 12-week exposure indicated adaptation of BEAS-2B cells to long-term stress. Together, the findings indicate that 3R4F TPM had a stronger effect on oxidative phosphorylation, gene expression and proteins involved in oxidative stress than TPM from the candidate modified-risk tobacco product THS2.2.
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http://dx.doi.org/10.1016/j.fct.2018.02.013DOI Listing
May 2018

Mechanistic Evaluation of the Impact of Smoking and Chronic Obstructive Pulmonary Disease on the Nasal Epithelium.

Clin Med Insights Circ Respir Pulm Med 2017 5;11:1179548417710928. Epub 2017 Jun 5.

Philip Morris Products SA and Research & Development (R&D), Philip Morris International, Neuchâtel, Switzerland.

Chronic obstructive pulmonary disease (COPD) is one of the major causes of chronic morbidity and mortality worldwide. The development of markers of COPD onset is hampered by the lack of accessibility to the primary target tissue, and there is a need to consider other sample sources as surrogates for biomarker research. Airborne toxicants pass through the nasal epithelium before reaching the lower airways, and the similarity with bronchial histology makes it an attractive surrogate for lower airways. In this work, we describe the transcriptomics findings from the nasal epithelia of subjects enrolled in a clinical study focusing on the identification of COPD biomarkers. Transcriptomic data were analyzed using the biological network approach that enabled us to pinpoint the biological processes affected in the upper respiratory tract in response to smoking and mild-to-moderate COPD. Our results indicated that nasal and lower airway immune responses were considerably different in COPD subjects and caution should be exercised when using upper airway samples as a surrogate for the lower airway. Nevertheless, the network approach described here could present a sensitive means of identifying smokers at risk of developing COPD.
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http://dx.doi.org/10.1177/1179548417710928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5466113PMC
June 2017

Alterations in Serum Polyunsaturated Fatty Acids and Eicosanoids in Patients with Mild to Moderate Chronic Obstructive Pulmonary Disease (COPD).

Int J Mol Sci 2016 Sep 20;17(9). Epub 2016 Sep 20.

Philip Morris International Research and Development, Philip Morris Products S.A. (Part of Philip Morris International Group of Companies), Quai Jeanrenaud 5, 2000 Neuchatel, Switzerland.

Smoking is a major risk factor for several diseases including chronic obstructive pulmonary disease (COPD). To better understand the systemic effects of cigarette smoke exposure and mild to moderate COPD-and to support future biomarker development-we profiled the serum lipidomes of healthy smokers, smokers with mild to moderate COPD (GOLD stages 1 and 2), former smokers, and never-smokers ( = 40 per group) (ClinicalTrials.gov registration: NCT01780298). Serum lipidome profiling was conducted with untargeted and targeted mass spectrometry-based lipidomics. Guided by weighted lipid co-expression network analysis, we identified three main trends comparing smokers, especially those with COPD, with non-smokers: a general increase in glycero(phospho)lipids, including triglycerols; changes in fatty acid desaturation (decrease in ω-3 polyunsaturated fatty acids, and an increase in monounsaturated fatty acids); and an imbalance in eicosanoids (increase in 11,12- and 14,15-DHETs (dihydroxyeicosatrienoic acids), and a decrease in 9- and 13-HODEs (hydroxyoctadecadienoic acids)). The lipidome profiles supported classification of study subjects as smokers or non-smokers, but were not sufficient to distinguish between smokers with and without COPD. Overall, our study yielded further insights into the complex interplay between smoke exposure, lung disease, and systemic alterations in serum lipid profiles.
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http://dx.doi.org/10.3390/ijms17091583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5037848PMC
September 2016

Alterations in the sputum proteome and transcriptome in smokers and early-stage COPD subjects.

J Proteomics 2015 Oct 22;128:306-20. Epub 2015 Aug 22.

Philip Morris International R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, 2000 Neuchâtel, Switzerland.

Chronic obstructive pulmonary disease (COPD) is one of the most prevalent lung diseases. Cigarette smoking is the main risk factor for COPD. In this parallel-group clinical study we investigated to what extent the transitions in a chronic-exposure-to-disease model are reflected in the proteome and cellular transcriptome of induced sputum samples. We selected 60 age- and gender-matched individuals for each of the four study groups: current asymptomatic smokers, smokers with early stage COPD, former smokers, and never smokers. The cell-free sputum supernatant was analyzed by quantitative proteomics and the cellular mRNA fraction by gene expression profiling. The sputum proteome of current smokers clearly reflected the common physiological responses to smoke exposure, including alterations in mucin/trefoil proteins and a prominent xenobiotic/oxidative stress response. The latter response also was observed in the transcriptome, which additionally demonstrated an immune-cell polarization change. The former smoker group showed nearly complete attenuation of these biological effects. Thirteen differentially abundant proteins between the COPD and the asymptomatic smoker group were identified including TIMP1, APOA1, C6orf58, and BPIFB1 (LPLUNC1). In summary, our study demonstrates that sputum profiling can capture the complex and reversible physiological response to cigarette smoke exposure, which appears to be only slightly modulated in early-stage COPD.
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http://dx.doi.org/10.1016/j.jprot.2015.08.009DOI Listing
October 2015

Systems toxicology approaches enable mechanistic comparison of spontaneous and cigarette smoke-related lung tumor development in the A/J mouse model.

Interdiscip Toxicol 2014 Jun 15;7(2):73-84. Epub 2014 Nov 15.

Biological Systems Research, Philip Morris International Research and Development, CH-2000 Neuchâtel, Switzerland.

The A/J mouse is highly susceptible to lung tumor induction and has been widely used as a screening model in carcinogenicity testing and chemoprevention studies. However, the A/J mouse model has several disadvantages. Most notably, it develops lung tumors spontaneously. Moreover, there is a considerable gap in our understanding of the underlying mechanisms of pulmonary chemical carcinogenesis in the A/J mouse. Therefore, we examined the differences between spontaneous and cigarette smoke-related lung tumors in the A/J mouse model using mRNA and microRNA (miRNA) profiling. Male A/J mice were exposed whole-body to mainstream cigarette smoke (MS) for 18 months. Gene expression interaction term analysis of lung tumors and surrounding non-tumorous parenchyma samples from animals that were exposed to either 300 mg/m(3) MS or sham-exposed to fresh air indicated significant differential expression of 296 genes. Ingenuity Pathway Analysis(®) (IPA(®)) indicated an overall suppression of the humoral immune response, which was accompanied by a disruption of sphingolipid and glycosaminoglycan metabolism and a deregulation of potentially oncogenic miRNA in tumors of MS-exposed A/J mice. Thus, we propose that MS exposure leads to severe perturbations in pathways essential for tumor recognition by the immune system, thereby potentiating the ability of tumor cells to escape from immune surveillance. Further, exposure to MS appeared to affect expression of miRNA, which have previously been implicated in carcinogenesis and are thought to contribute to tumor progression. Finally, we identified a 50-gene expression signature and show its utility in distinguishing between cigarette smoke-related and spontaneous lung tumors.
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http://dx.doi.org/10.2478/intox-2014-0010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427718PMC
June 2014

Lung cancer biomarkers for the assessment of modified risk tobacco products: an oxidative stress perspective.

Biomarkers 2013 May 27;18(3):183-95. Epub 2013 Mar 27.

British American Tobacco, Southampton, UK.

Manufacturers have developed prototype cigarettes yielding reduced levels of some tobacco smoke toxicants, when tested using laboratory machine smoking under standardised conditions. For the scientific assessment of modified risk tobacco products, tests that offer objective, reproducible data, which can be obtained in a much shorter time than the requirements of conventional epidemiology are needed. In this review, we consider whether biomarkers of biological effect related to oxidative stress can be used in this role. Based on published data, urinary 8-oxo-7,8-dihydro-2-deoxyguanosine, thymidine glycol, F2-isoprostanes, serum dehydroascorbic acid to ascorbic acid ratio and carotenoid concentrations show promise, while 4-hydroxynonenal requires further qualification.
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http://dx.doi.org/10.3109/1354750X.2013.777116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3667677PMC
May 2013

MicroRNAs as potential biomarkers of smoking-related diseases.

Biomark Med 2012 Oct;6(5):671-84

British American Tobacco, Group Research & Development, Southampton, Hampshire SO15 8TL, UK.

MicroRNAs (miRNAs) comprise a family of small, endogenous, noncoding functional RNA molecules that have emerged as key post-transcriptional regulators of gene expression. They inhibit the translation of proteins from mRNA or promote its degradation. Aberrant miRNA expression has been linked to various human diseases and measurement can differentiate between normal and diseased tissue. Expression is tissue-specific and any changes in miRNA expression within a tissue type can be correlated with disease status. Altered miRNA expression has been reported in the smoking-related diseases cancer, chronic obstructive pulmonary disease and cardiovascular disease. Additionally, miRNAs are thought to have vital roles in inflammatory cell differentiation and regulation. miRNAs might, therefore, be useful biomarkers for early detection of disease-related molecular and genetic changes. In this review, we summarize the available scientific evidence for the potential of miRNAs as biomarkers of smoking-related diseases. Studies should be carried out to identify the miRNAs most relevant to specific diseases.
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http://dx.doi.org/10.2217/bmm.12.50DOI Listing
October 2012

Gene expression profiling of human alveolar macrophages of phenotypically normal smokers and nonsmokers reveals a previously unrecognized subset of genes modulated by cigarette smoking.

J Mol Med (Berl) 2006 Apr 7;84(4):318-28. Epub 2006 Mar 7.

Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA.

Cigarette smoking is the leading cause of the respiratory diseases collectively known as chronic obstructive pulmonary disease (COPD). While the pathogenesis of COPD is complex, there is abundant evidence that alveolar macrophages (AM) play an important role. Based on the concept that COPD is a slow-progressing disorder likely involving multiple mediators released by AM activated by cigarette smoke, the present study focuses on the identification of previously unrecognized genes that may be linked to early events in the molecular pathogenesis of COPD, as opposed to factors associated with the presence of disease. To accomplish this, microarray analysis using Affymetrix microarrays was used to carry out an unbiased survey of the differences in gene expression profiles in the AM of phenotypically normal, approximately 20 pack-year smokers compared to healthy nonsmokers. Although smoking did not alter the global gene expression pattern of AM, 75 genes were modulated by smoking, with 40 genes up-regulated and 35 down-regulated in the AM of smokers compared to nonsmokers. Most of these genes belong to the functional categories of immune/inflammatory response, cell adhesion and extracellular matrix, proteolysis and antiproteolysis, lysosomal function, antioxidant-related function, signal transduction, and regulation of transcription. Of these 75 genes, 69 have not been previously recognized to be up- or down-regulated in AM in association with smoking or COPD, including genes coding for proteins belonging to all of the above categories, and others belonging to various functional categories or of unknown function. These observations suggest that gene expression responses of AM associated with the stress of cigarette smoking are more complex than previously thought, and offer a variety of new insights into the complex pathogenesis of smoking-induced lung diseases.
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http://dx.doi.org/10.1007/s00109-005-0008-2DOI Listing
April 2006

Similarity of gene expression patterns in human alveolar macrophages in response to Pseudomonas aeruginosa and Burkholderia cepacia.

Infect Immun 2005 Aug;73(8):5262-8

Department of Genetic Medicine, Weill Medical College of Cornell University, 515 East 71st Street, S-1000, New York, NY 10021, USA.

To determine if differences in the severity of pulmonary infection in cystic fibrosis seen with late isolates of Pseudomonas aeruginosa and Burkholderia cepacia are associated with differences in the initial response of alveolar macrophages (AM) to these pathogens, we assessed gene expression changes in human AM in response to infection with a laboratory strain, early and late clinical isolates of P. aeruginosa, and B. cepacia. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays, following exposure to laboratory P. aeruginosa strain PAK, showed significant (P < 0.01) >2.5-fold upregulation of 42 genes and >2.5-fold downregulation of 45 genes. The majority of the changes in gene expression involved genes as part of inflammatory pathways and signaling systems. Interestingly, similar responses were observed following exposure of AM to early and late clinical isolates of P. aeruginosa, as well as with B. cepacia, suggesting that the more severe clinical outcome of infections with late clinical isolates of P. aeruginosa or with B. cepacia cannot be explained by differences in the early interactions of these organisms with the human AM, as reflected by the similarity of gene expression changes in response to exposure of AM to these pathogens.
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http://dx.doi.org/10.1128/IAI.73.8.5262-5268.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1201277PMC
August 2005

Trans-splicing repair of CD40 ligand deficiency results in naturally regulated correction of a mouse model of hyper-IgM X-linked immunodeficiency.

Nat Med 2004 Aug 25;10(8):835-41. Epub 2004 Jul 25.

Department of Genetic Medicine, Weill Medical College of Cornell University, New York, New York, USA.

X-linked immunodeficiency with hyper-IgM (HIGM1), characterized by failure of immunoglobulin isotype switching, is caused by mutations of the CD40 ligand (CD40L), which is normally expressed on activated CD4(+) T cells. As constitutive expression of CD40L induces lymphomas, we corrected the mutation while preserving the natural regulation of CD40L using pre-mRNA trans-splicing. Bone marrow from mice lacking CD40L was modified with a lentivirus trans-splicer encoding the normal CD40L exons 2-5 and was administered to syngenic CD40L-knockout mice. Recipient mice had corrected CD40L mRNA, antigen-specific IgG1 responses to keyhole limpet hemocyanin immunization, regulated CD4(+) T-cell CD40L expression after CD3 stimulation in primary and secondary transplanted mice, attenuation of Pneumocystis carinii pneumonia, and no evidence of lymphoproliferative disease over 1 year. Thus, HIGM1 can be corrected by CD40L trans-splicing, leading to functional correction of the genetic defect without the adverse consequences of unregulated expression of the CD40L gene.
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http://dx.doi.org/10.1038/nm1086DOI Listing
August 2004

TGFbeta1 activates c-Jun and Erk1 via alphaVbeta6 integrin.

Mol Cancer 2003 Sep 23;2:33. Epub 2003 Sep 23.

1Department of Dermatology, Weill Medical College of Cornell University, 1300 York Avenue, New York, New York, 10021, USA.

Transforming growth factor beta (TGFbeta) plays an important role in animal development and many cellular processes. A variety of cellular functions that are required for tumor metastasis are controlled by integrins, a family of cell adhesion receptors. Overexpression of alphaVbeta6 integrin is associated with lymph node metastasis of gastric carcinomas. It has been demonstrated that a full TGFbeta1 signal requires both alphaVbeta6 integrin and SMAD pathway. TGFbeta1 binds to alphaVbeta6 via the DLXXL motif, a freely accessible amino acid sequence in the mature form of TGFbeta1. Binding of mature TGFbeta1 to alphaVbeta6 leads to immobilization and tyrosine phosphorylation of proteins, which are associated with focal adhesions, a hallmark of integrin-mediated signal transduction. Here, we show that binding of mature TGFbeta1 recruits the mitogen-activated protein kinase kinase kinase 1 (MEKK1), a mediator of c-Jun activation, and the extracellular signaling-regulated kinase-1 (Erk1) to focal adhesions. In addition, the p21-activated kinase 1 (PAK1) is associated with focal adhesions and differentially phosphorylated upon TGFbeta1 stimulation. We conclude that TGFbeta1 activates c-Jun via the MEKK1/p38 MAP kinase pathway and influences cytoskeletal organization. These finding may provide a link between TGFbeta1 and the metastatic behavior of cancers.
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http://dx.doi.org/10.1186/1476-4598-2-33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC222989PMC
September 2003

Analysis of fetal DNA in the maternal venous blood for abnormalities of chromosomes 13, 16, 18 and 21 in first-trimester spontaneous miscarriage.

J Obstet Gynaecol 2003 May;23(3):228-32

Coombe Women's Hospital, Dublin, Ireland.

We investigated whether fetal DNA from the maternal blood can be used to diagnose abnormalities of chromosomes 13, 16, 18 and 21 in spontaneous miscarriage. Venous blood was obtained from 50 women with a diagnosis of spontaneous miscarriage. The evacuated products of the uterus were also sampled. DNA was extracted from both blood and tissue samples. Polymerase chain reaction (PCR) was performed using microsatellite markers for selected chromosomes. A novel technique using a capillary sequencer was employed. The electrophoretogram created was analysed using GeneScan software. The result obtained from the maternal blood samples was compared to the result obtained from uterine samples. Forty-two of the 50 (84%) cases were found to have a trisomy of one or more of the chromosomes tested on analysis of maternal blood. Compared to the overall result obtained on the uterine products of conception, the sensitivity was 97.6 and specificity 1.0. Trisomy 16 was the most common abnormality detected. These results suggest that the incidence of genetic abnormalities complicating miscarriage may be higher than suspected hitherto.
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http://dx.doi.org/10.1080/0144361031000098307DOI Listing
May 2003

Variability of antioxidant-related gene expression in the airway epithelium of cigarette smokers.

Am J Respir Cell Mol Biol 2003 Sep 17;29(3 Pt 1):331-43. Epub 2003 Apr 17.

Belfer Gene Therapy Core Facility, Department of Genetic Medicine, Weill Medical College of Cornell University, New York, New York 10021, USA.

Cigarette smoking is the major risk factor for developing chronic bronchitis, yet only 15-20% of smokers develop this disorder. Because oxidants are the major mechanism of smoking-induced airway damage, we hypothesized that smoking is associated with upregulation of various antioxidant-related genes in the airway epithelium, but the magnitude of the response shows high inter-individual variability. Microarray analysis was used to assess levels of expression of 44 antioxidant-related genes in four categories (catalase/superoxide dismutase family; glutathione metabolism; redox balance; and pentose phosphate cycle) in bronchoscopy-obtained airway epithelium of matched cohorts (13 current smokers, 9 nonsmokers), none of whom had lung disease. There was minimal variation in gene expression levels within the same individual (right versus left lung or over time), but significant upregulation of 16/44 antioxidant-related genes in smoker epithelium compared with nonsmokers. Subgroups of smokers were identified with clusters of expression levels of antioxidant-related genes. We propose that the antioxidant-related genes demonstrating the most variability in the level of expression in smokers may be useful genetic markers in epidemiologic studies assessing susceptibility to smoking-induced chronic bronchitis.
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http://dx.doi.org/10.1165/rcmb.2002-0321OCDOI Listing
September 2003

Monoallelic up-regulation of the imprinted H19 gene in airway epithelium of phenotypically normal cigarette smokers.

Cancer Res 2003 Apr;63(7):1475-82

Division of Pulmonary and Critical Care Medicine, Weill Medical College of Cornell University, New York, New York 10021, USA.

H19, a paternally imprinted gene, is postulated to have regulatory functions in normal development and oncogenesis. Loss of imprinting (LOI) of H19 is observed in human malignancies, including lung cancer. Microarray assessment of gene expression patterns in airway epithelium of healthy 20 pack-year smokers versus nonsmokers revealed that smokers have dramatically elevated H19 RNA levels without alteration of expression of other imprinted genes. Interestingly, the up-regulation of H19 was not attributable to LOI, i.e., expression of H19 in smokers was monoallelic. These observations suggest that cigarette smoking initially induces up-regulation of the active H19 allele and that there is likely progression to LOI as the burden of smoking increases and as the epithelium undergoes transition from normal to neoplastic. Overexpression and eventual LOI of H19 may represent early markers in the progression of airway epithelium toward lung cancer.
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April 2003