Philip Morris Research Laboratories GmbH

Köln, Germany

Philip Morris Research Laboratories GmbH

Köln, Germany
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Steffen Y.,Philip Morris Research Laboratories GmbH | Vuillaume G.,Philip Morris Products SA | Stolle K.,Philip Morris Research Laboratories GmbH | Roewer K.,Philip Morris Research Laboratories GmbH | And 4 more authors.
Nitric Oxide - Biology and Chemistry | Year: 2012

The ubiquitous free radical nitric oxide (NO) plays an important role in many biological processes, including the regulation of both vascular tone and inflammatory response; however, its role in the effects of cigarette smoke exposure on atherosclerosis remains unclear. Our aim was to study the mechanisms of NO regulation in endothelial cells in response to cigarette smoke exposure in vitro. Using human umbilical vein endothelial cells (HUVEC), we have demonstrated that combining non-toxic concentrations of cigarette smoke bubbled through PBS (smoke-bubbled PBS [sbPBS]) with native LDL (nLDL) significantly reduces the amount of bioavailable NO. The effect is comparable to that seen with oxidized LDL (oxLDL), but has not been seen with sbPBS or nLDL alone. Mechanistic investigations showed that the combination of sbPBS + nLDL did not reduce the amount of endothelial nitric oxide synthase (eNOS), but did inhibit its enzymatic activity. Concomitantly, both sbPBS + nLDL and oxLDL significantly increased the production of reactive oxygen species (ROS) in the form of superoxide anions (O2-) and peroxynitrite (ONOO-) in HUVEC. Selective inhibition of NADPH oxidase prevented this response. Incubation of sbPBS + nLDL revealed the formation of 7-ketocholesterol (7-KC) and 7-hydroxycholesterol, which are indicators for oxidative modification of LDL. This could explain the reported increase in circulatory levels of oxLDL in smokers. Our results suggest that reduction of functional NO in response to a combination of sbPBS + nLDL is secondary to both reduction of eNOS activity and stimulation of NADPH oxidase activity. Because sbPBS alone showed no effect on eNOS activity or ROS formation, nLDL should be included in cigarette-smoke-related mechanistic in vitro experiments on endothelial cells to be more reflective of the clinical situation. © 2012 Elsevier Inc. All rights reserved.

Martin F.,Philip Morris Products S.A. | Thomson T.M.,Selventa | Sewer A.,Philip Morris Products S.A. | Drubin D.A.,Selventa | And 5 more authors.
BMC Systems Biology | Year: 2012

Background: High-throughput measurement technologies produce data sets that have the potential to elucidate the biological impact of disease, drug treatment, and environmental agents on humans. The scientific community faces an ongoing challenge in the analysis of these rich data sources to more accurately characterize biological processes that have been perturbed at the mechanistic level. Here, a new approach is built on previous methodologies in which high-throughput data was interpreted using prior biological knowledge of cause and effect relationships. These relationships are structured into network models that describe specific biological processes, such as inflammatory signaling or cell cycle progression. This enables quantitative assessment of network perturbation in response to a given stimulus.Results: Four complementary methods were devised to quantify treatment-induced activity changes in processes described by network models. In addition, companion statistics were developed to qualify significance and specificity of the results. This approach is called Network Perturbation Amplitude (NPA) scoring because the amplitudes of treatment-induced perturbations are computed for biological network models. The NPA methods were tested on two transcriptomic data sets: normal human bronchial epithelial (NHBE) cells treated with the pro-inflammatory signaling mediator TNFα, and HCT116 colon cancer cells treated with the CDK cell cycle inhibitor R547. Each data set was scored against network models representing different aspects of inflammatory signaling and cell cycle progression, and these scores were compared with independent measures of pathway activity in NHBE cells to verify the approach. The NPA scoring method successfully quantified the amplitude of TNFα-induced perturbation for each network model when compared against NF-κB nuclear localization and cell number. In addition, the degree and specificity to which CDK-inhibition affected cell cycle and inflammatory signaling were meaningfully determined.Conclusions: The NPA scoring method leverages high-throughput measurements and a priori literature-derived knowledge in the form of network models to characterize the activity change for a broad collection of biological processes at high-resolution. Applications of this framework include comparative assessment of the biological impact caused by environmental factors, toxic substances, or drug treatments. © 2012 Martin et al.; licensee BioMed Central Ltd.

Piade J.-J.,Philip Morris Products SA | Roemer E.,Philip Morris Products SA | Dempsey R.,Philip Morris Products SA | Weiler H.,Philip Morris Research Laboratories GmbH | And 4 more authors.
Regulatory Toxicology and Pharmacology | Year: 2014

A typical Indonesian kretek cigarette brand and an experimental kretek reference cigarette were compared to the reference cigarette 2R4F in two 90-day inhalation studies. Male and female rats were exposed nose-only to mainstream smoke for 6 hours daily, for 90 consecutive days. Biological endpoints were assessed according to OECD guideline 413, with special emphasis on respiratory tract histopathology and on lung inflammation (broncho-alveolar lavage fluid levels of neutrophils, macrophages and lymphocytes). Histopathological alterations included: in the nose, hyperplasia and squamous metaplasia of the respiratory epithelium and squamous metaplasia and atrophy of the olfactory epithelium; in the larynx, epithelial squamous metaplasia and hyperplasia; in the lungs, accumulation of macrophages in alveoli and goblet cell hyperplasia in bronchial epithelium. The findings were qualitatively consistent with observations from previous similar studies on conventional cigarettes. Compared to 2R4F cigarette, however, kretek smoke exposure was associated with a pronounced attenuation of pulmonary inflammation and less severe histopathological changes in the respiratory tract. Neutrophilic inflammation was also significantly lower (>70%). These results are consistent with the observations made on smoke chemistry and in vitro toxicology. They do not support any increased toxicity of the smoke of kretek cigarettes compared to conventional American-blended cigarettes. © 2014 Elsevier Inc.

Roemer E.,Philip Morris Products SA | Dempsey R.,Philip Morris Products SA | Hirter J.,Philip Morris Products SA | Deger Evans A.,Philip Morris Products SA | And 4 more authors.
Regulatory Toxicology and Pharmacology | Year: 2014

Mainstream smoke (MS) from experimental kretek cigarettes with three ingredient mixes at low (typical use level) and high (2.5 or 3 times that level) inclusion rates was compared to a control kretek cigarette of identical construction (cloves and humectants), but without the addition of ingredients. 350 ingredients, commonly used in various combinations and in a limited number in a given brand in the manufacture of marketed kretek cigarettes were assessed. The MS composition of the kretek cigarettes was characterized by a comprehensive set of analytes (55 smoke constituents). Furthermore, the smoke was assessed in vitro for its cytotoxicity in the Neutral Red Uptake assay (particle phase and gas/vapor phase separately) in mouse embryo BALB/c 3T3 cells, and for mutagenicity/genotoxicity in the Salmonella typhimurium reverse mutation assay and the mammalian cell mouse lymphoma TK assay in L5178Y cells, the latter with and without metabolic activation. There were some statistically significant differences in the yield of smoke constituents (increases as well as decreases, nearly all of them less than ±20%) as a result of the addition of the ingredient mixes. However, the addition of the three different mixes of ingredients to the experimental kreteks did not change the in vitro cytotoxicity and mutagenicity/genotoxicity of the smoke, when compared to the control kretek cigarette. © 2014 Elsevier Inc.

Roemer E.,Philip Morris Products SA | Dempsey R.,Philip Morris Products SA | Lawless-Pyne J.,PT HM Sampoerna Tbk | Lukman S.,PT HM Sampoerna Tbk | And 4 more authors.
Regulatory Toxicology and Pharmacology | Year: 2014

The smoke chemistry and in vitro toxicity of mainstream smoke (MS) was investigated in American-blended cigarettes with or without the addition of 2.5%, 5% or 10% eugenol to the tobacco and in Indonesian-blended cigarettes with and without the addition of cloves, cloves extracted with hot ethanol, and extracted cloves replenished with eugenol or clove oil. The addition of eugenol reduced the concentration of nearly all toxicants measured in MS as well as the in vitro cytotoxicity of the gas/vapor phase. Reductions were also seen in bacterial mutagenicity of the total particulate matter (TPM) assessed by the Ames Assay. The addition of extracted cloves led to increases and decreases of toxicant concentrations in MS. Replenishment with eugenol or clove oil decreased the toxicant concentrations; with most smoke constituent concentrations reduced below the concentration found in tobacco-only cigarettes. Cytotoxicity of the TPM was not affected by the clove preparations. However, GVP cytotoxicity was reduced (untreated cloves showing the highest reductions). Mutagenicity of TPM was decreased by the clove preparations. Mechanisms for the reductions, (up to 40%), are most likely due to dilution effects by eugenol, changed burning characteristics of the tobacco, and free radical scavenging by eugenol. © 2014 Elsevier Inc.

Poussin C.,Philip Morris Products S.A. | Gallitz I.,Philip Morris Research Laboratories GmbH | Schlage W.K.,Philip Morris Research Laboratories GmbH | Steffen Y.,Philip Morris Products S.A. | And 5 more authors.
Toxicology in Vitro | Year: 2014

The adhesion of monocytic cells to the "dysfunctional" endothelium constitutes a critical step in the initiation of atherosclerosis. Cigarette smoke (CS) has been shown to contribute to this process, the complex mechanism of which still needs to be unraveled. We developed an in vitro adhesion assay to investigate the CS-induced adhesion of monocytic MM6 cells to human umbilical vein endothelial cells (HUVECs) following exposure to an aqueous CS extract (smoke-bubbled phosphate buffered saline: sbPBS), reasoning that in vivo monocytes and endothelial cells are exposed primarily to soluble constituents from inhaled CS absorbed through the lung alveolar wall. MM6 cell adhesion was increased exclusively by the conditioned medium from sbPBS-exposed MM6 cells, not by direct sbPBS exposure of the HUVECs within a range of sbPBS doses. Using a transcriptomics approach followed by confirmation experiments, we identified different exposure effects on both cell types and a key mechanism by which sbPBS promoted the adhesion of MM6 cells to HUVECs. While sbPBS provoked a strong oxidative stress response in both cell types, the expression of E-selectin, VCAM-1 and ICAM-1, responsible for the adhesion of MM6 cells to HUVECs, was induced in the latter through a proinflammatory paracrine effect. We confirmed that this effect was driven mainly by TNFα produced by MM6 cells exposed to sbPBS. In conclusion, we have elucidated an indirect mechanism by which sbPBS increases the adhesion of monocytic cells to endothelial cells in this in vitro assay that was designed for tobacco product risk assessment while mimicking the in vivo exposure conditions as closely as possible. © 2014 The Authors.

Gebe S.,Philip Morris Research Laboratories GmbH | Dieh S.,Philip Morris Research Laboratories GmbH | Pype J.,Philip Morris Research Laboratories bvba | Friedrichs B.,Philip Morris Research Laboratories GmbH | And 6 more authors.
Toxicological Sciences | Year: 2010

Cigarette smoke (CS) imposes a strong oxidative burden on exposed tissues resulting in a severely disturbed oxidant/antioxidant balance, which in the context of chronic exposure is assumed to be a key contributor to CS-related diseases. Because of its emerging central role in orchestrating the general cellular antioxidant response, the pathway leading to the activation of the transcription factor Nrf2 has received mounting attention over the past decade in investigations aimed at elucidating CS-induced pathophysiological mechanisms. To comprehensively characterize the impact of Nrf2 in acute and subchronic smoking scenarios, Nrf 2-/- mice and their wild-type (wt) ICR littermates were exposed to either ambient air (sham exposure) or one of three doses of CS for up to 5 months, with two postexposure endpoints of 1 and 13 days. The lungs of the mice were monitored for transcriptomic changes on a genome-wide level, which confirmed an impaired expression of antioxidant and phase 2-related genes in CS-exposed Nrf 2-/- mice. Importantly, in comparison to wt mice, an attenuated cell cycle/mitotic response and intensified stress gene expression pattern were observed in exposed Nrf 2-/- mice, which was paralleled by clear dose-dependent effects on alveolar destruction and impaired lung function. In contrast, the inflammation-related transcriptional response and scores for various bronchioalveolar inflammation parameters were qualitatively and quantitatively similar in CS-exposed mice of both genotypes. Taken together, these results confirm the protective nature of Nrf2 in oxidative stress scenarios and suggest that the enhanced emphysematous phenotype exhibited by CS-exposed Nrf 2-/- mice is more likely caused by an imbalance in cell loss and regeneration than by increased inflammation. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

Friedrichs B.,Philip Morris Research Laboratories GmbH | Neumann U.,Philip Morris Research Laboratories GmbH | Schuller J.,Philip Morris Research Laboratories GmbH | Peck M.J.,Philip Morris Products S.A.
Toxicology in Vitro | Year: 2014

In vitro treatment of human peripheral blood neutrophils from smokers and non-smokers with an aqueous cigarette smoke (CS) extract resulted in a concentration-dependent increase in surface expression of CD11b and CD66b and a corresponding decrease of CD62L, together with a concentration-dependent release of MMP-8, MMP-9, and lactoferrin, indicating considerable activation and degranulation. However, the burst response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) was unchanged in CS-stimulated neutrophils from both smokers and non-smokers. When supernatants from CS-treated monocytic MonoMac-6 (MM6) cells were used for activation of neutrophils, concentration-dependent changes in surface marker expression, granule protein release, and the oxidative burst response to fMLP were observed, again with no major differences between smokers and non-smokers. CS-treated MM6 cells released significant amounts of IL-8 and TNF-α into the culture supernatant. However, antibody blocking experiments showed that only TNF-α mediated the increased burst response in neutrophils. These data show that, in the presence of secondary cells, CS is able to prime neutrophils for an increased burst response to fMLP which is mediated by TNF-α, released from the secondary cells in response to CS. Following stimulation with priming agents, peripheral blood neutrophils from healthy smokers show an equal burst response compared to those from non-smokers. © 2014.

Knorr A.,Philip Morris Products S.A. | Monge A.,Philip Morris Products S.A. | Stueber M.,Philip Morris Research Laboratories GmbH | Stratmann A.,Philip Morris Research Laboratories GmbH | And 3 more authors.
Analytical Chemistry | Year: 2013

Compound identification is widely recognized as a major bottleneck for modern metabolomic approaches and high-throughput nontargeted characterization of complex matrices. To tackle this challenge, an automated platform entitled computer-assisted structure identification (CASI) was designed and developed in order to accelerate and standardize the identification of compound structures. In the first step of the process, CASI automatically searches mass spectral libraries for matches using a NIST MS Search algorithm, which proposes structural candidates for experimental spectra from two-dimensional gas chromatography with time-of-flight mass spectrometry (GC × GC-TOF-MS) measurements, each with an associated match factor. Next, quantitative structure-property relationship (QSPR) models implemented in CASI predict three specific parameters to enhance the confidence for correct compound identification, which were Kovats Index (KI) for the first dimension (1D) separation, relative retention time for the second dimension separation (2DrelRT) and boiling point (BP). In order to reduce the impact of chromatographic variability on the second dimension retention time, a concept based upon hypothetical reference points from linear regressions of a deuterated n-alkanes reference system was introduced, providing a more stable relative retention time measurement. Predicted values for KI and 2DrelRT were calculated and matched with experimentally derived values. Boiling points derived from 1D separations were matched with predicted boiling points, calculated from the chemical structures of the candidates. As a last step, CASI combines the NIST MS Search match factors (NIST MF) with up to three predicted parameter matches from the QSPR models to generate a combined CASI Score representing the measure of confidence for the identification. Threshold values were applied to the CASI Scores assigned to proposed structures, which improved the accuracy for the classification of true/false positives and true/false negatives. Results for the identification of compounds have been validated, and it has been demonstrated that identification using CASI is more accurate than using NIST MS Search alone. CASI is an easily accessible web-interfaced software platform which represents an innovative, high-throughput system that allows fast and accurate identification of constituents in complex matrices, such as those requiring 2D separation techniques. © 2013 American Chemical Society.

PubMed | Philip Morris Research Laboratories GmbH and Philip Morris Products S.A.
Type: | Journal: Toxicology letters | Year: 2016

Heterocyclic aromatic amines (HAAs) rank among the strongest known mutagens. Approximately 30 HAAs have been found in cooked foods (broiled, fried, and grilled) and several HAAs have been characterized as animal carcinogens. Nine HAAs have also been reported to be constituents of cigarette smoke (CS) raising concerns that HAAs might contribute significantly to the known carcinogenicity of CS. As HAAs are found predominantly in the total particulate matter (TPM) of CS, an improved method for the quantification of HAAs in TPM is reported allowing detection and quantification of 8 HAAs in a single run. The mutagenic potency of these HAAs and that of TPM from the reference cigarette 2R4F was determined in the Salmonella Reverse Mutation Assay (Ames assay) with tester strain TA98 and a metabolic activation system. The 8 HAAs, when applied together in the Ames assay, showed a clear sub-additive response. Likewise, the combination of HAAs and TPM, if at all, gave rise to a slight sub-additive response. In both cases, however, the sub-additive response in the Ames assay was observed at HAA doses that are far above the amounts found in CS. The contribution of the individual HAAs to the total mutagenic activity of TPM was calculated and experimentally confirmed to be approximately 1% of the total mutagenic activity. Thus, HAAs do not contribute significantly to the bacterial in vitro mutagenicity of CS TPM.

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