7-month systems toxicology inhalation / cessation study with pMRTP in C57BL6 mice

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Chronic obstructive pulmonary disease (COPD) is a major cause of chronic morbidity and mortality worldwide. Cigarette smoking and both indoor and outdoor air pollution are important risk factors contributing to the pathogenesis of this preventable, complex pulmonary disorder characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and lung to noxious particles or gases.

PMI is developing products with the potential to reduce individual risk and population harm (Modified Risk Tobacco Products (MRTPs) also referred to by PMI as Reduced Risk Products (RRPs)).

Utilizing a switching study concept in a previously established murine model of COPD, C57BL/6 mice, allows to compare the degree of similarity between (i) switching to a prototypic MRTP and (ii) complete smoking cessation. This is studied in comparison to continuous smoke exposure in order to identify endpoints and biological network perturbations that are:

  • neither reversed by cessation nor switching
  • reversed by both cessation and switching
  • only reversed by cessation


Female C57BL/6 mice were exposed to diluted mainstream aerosols from 3R4F or pMRTP for 4 hours per day, 5 days a week, for up to 7 months. The exposure concentrations for the 3R4F and pMRTP aerosols were matched for nicotine content, with a target nicotine concentration of 34.4 µg/l (equivalent to 750 mg/m3 in the 3R4F aerosol).

Uptake of the aerosols was confirmed by monitoring blood carboxyhemoglobin and secreted urinary nicotine metabolites.

At the scheduled dissection time points (months 1, 2, 3, 4, 5, and 7), the animals were examined for multiple parameters to comprehensively assess the development of COPD, including pulmonary inflammation measurements, an assessment of pulmonary function, histopathological examination, and the determination of molecular changes throughout the exposure period by transcriptomics, proteomics and lipidomics.

Study Design and Conduct

Study design

The study design included five groups of female C57BL/6 mice:

  1. Sham (exposed to fresh air)
  2. 3R4F (exposed to mainstream smoke from the reference cigarette 3R4F, 750 mg total particulate matter (TPM)/m3 equivalent to 34.4 µg nicotine/l)
  3. pMRTP (exposed to mainstream aerosol from a pMRTP in which the nicotine level was matched to that of 3R4F)
  4. (Exposure) Cessation
  5. Switch (to pMRTP)

Choice of switching time point

The time point for switching was chosen based on the results of a previously conducted inhalation study on C57BL/6 indicating that lung inflammation, emphysema, and changed pulmonary function were apparent after 2 months of exposure to 3R4F1

A systems toxicology study

The combined assessment of classical toxicological end points with additional transcriptomics, proteomics, and lipidomics investigations in one study that includes a comparably small extra cohort of animals allows for the simultaneous investigation of a multitude of mechanistic parameters, biomarkers etc. that can be directly correlated with the toxicological outcomes, thereby avoiding the need for separate mechanistic studies with a higher number of animals including those for bridging between studies, confirmation of biological outcomes, positive and negative controls, etc.

Choice of gender

Female mice were chosen because of a possible increased susceptibility to develop emphysema2.

Detailed study design

The allocation of mice to the treatment groups, endpoint class, and time-points is shown below. The numbers in the table indicate the number of mice (i.e. biological replicates) per group.

C57BL6 mice 7 months (pMRTP) Results

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Publications and Reports

Martin F. et al. Human & experimental toxicology 34: 1200-1211 (2015)

Identification of gene expression signature for cigarette smoke exposure response--from man to mouse.

Gene expression profiling data can be used in toxicology to assess both the level and impact of toxicant exposure, aligned with a vision of 21st toxicology. Here, we present a whole blood derived gene signature that can distinguish current smokers from either non-smokers or former smokers with high specificity and sensitivity. Such a signature that can be measured in a surrogate tissue (whole blood) may help in monitoring smoking exposure as well as discontinuation of exposure when the primarily impacted tissue (e.g. lung) is not readily accessible. The signature consisted of LRRN3, SASH1, PALLD, RGL1, TNFRSF17, CDKN1C, IGJ, RRM2, ID3, SERPING1 and FUCA1. Several members of this signature have been previously described in the context of smoking. The signature translated well across species and could distinguish mice that were exposed to cigarette smoke from ones exposed to air only or had been withdrawn from cigarette smoke exposure. Finally, the small signature of only 11 genes could be converted into a PCR-based assay that could serve as a marker to monitor compliance with a smoking abstinence protocol.

Titz, B. et al. Toxicological sciences 149: 441-457 (2016)

Effects of Cigarette Smoke, Cessation, and Switching to Two Heat-Not-Burn Tobacco Products on Lung Lipid Metabolism in C57BL/6 and Apoe-/- Mice-An Integrative Systems Toxicology Analysis.

The impact of cigarette smoke (CS), a major cause of lung diseases, on the composition and metabolism of lung lipids is incompletely understood. Here, we integrated quantitative lipidomics and proteomics to investigate exposure effects on lung lipid metabolism in a C57BL/6 and an Apolipoprotein E-deficient (Apoe-/-) mouse study. In these studies, mice were exposed to high concentrations of 3R4F reference CS, aerosol from potential modified risk tobacco products (MRTPs) or filtered air (Sham) for up to 8 months. The two assessed MRTPs, the prototypical MRTP for C57BL/6 mice and the Tobacco Heating System 2.2 for Apoe-/- mice, utilize "heat-not-burn" technologies and were each matched in nicotine concentrations to the 3R4F CS. After 2 months of CS exposure, some groups were either switched to the MRTP or underwent cessation. In both mouse strains, CS strongly affected several categories of lung lipids and lipid-related proteins. Candidate surfactant lipids, surfactant proteins and surfactant metabolizing proteins were increased. Inflammatory eicosanoids, their metabolic enzymes and several ceramide classes were elevated. Overall, CS induced a coordinated lipid response controlled by transcription regulators such as SREBP proteins and supported by other metabolic adaptations. In contrast, most of these changes were absent in the mice exposed to the potential MRTPs, in the cessation group, and the switching group. Our findings demonstrate the complex biological response of the lungs to CS exposure and support the benefits of cessation or switching to a heat-not-burn product using a design such as those employed in this study.

Phillips, B. et al. Food and chemical toxicology, 80: 328-345 (2015)

A 7-month cigarette smoke inhalation study in C57BL/6 mice demonstrates reduced lung inflammation and emphysema following smoking cessation or aerosol exposure from a prototypic modified risk tobacco product.

Modified risk tobacco products (MRTP) are designed to reduce smoking-related health risks. A murine model of chronic obstructive pulmonary disease (COPD) was applied to investigate classical toxicology end points plus systems toxicology (transcriptomics and proteomics). C57BL/6 mice were exposed to conventional cigarette smoke (3R4F), fresh air (sham), or a prototypic MRTP (pMRTP) aerosol for up to 7 months, including a cessation group and a switching-to-pMRTP group (2 months of 3R4F exposure followed by fresh air or pMRTP for up to 5 months respectively). 3R4F smoke induced the typical adaptive changes in the airways, as well as inflammation in the lung, associated with emphysematous changes (impaired pulmonary function and alveolar damage). At nicotine-matched exposure concentrations of pMRTP aerosol, no signs of lung inflammation and emphysema were observed. Both the cessation and switching groups showed a similar reversal of inflammatory responses and no progression of initial emphysematous changes. A significant impact on biological processes, including COPD-related inflammation, apoptosis, and proliferation, was identified in 3R4Fexposed, but not in pMRTP-exposed lungs. Smoking cessation or switching reduced these perturbations to near sham-exposed levels. In conclusion, the mouse model indicated retarded disease progression upon cessation or switching to pMRTP which alone had no adverse effects. Full text including Supplement

Ansari, S. et al. Scientific Data 3: 150077 (2016)

Comprehensive systems biology analysis of a 7-month cigarette smoke inhalation study in C57BL/6 mice.

Smoking of combustible cigarettes has a major impact on human health. Using a systems toxicology approach in a model of chronic obstructive pulmonary disease (C57BL/6 mice), we assessed the health consequences in mice of an aerosol derived from a prototype modified risk tobacco product (pMRTP) as compared to conventional cigarettes. We investigated physiological and histological endpoints in parallel with transcriptomics, lipidomics, and proteomics profiles in mice exposed to a reference cigarette (3R4F) smoke or a pMRTP aerosol for up to 7 months. We also included a cessation group and a switching-to-pMRTP group (after 2 months of 3R4F exposure) in addition to the control (fresh air-exposed) group, to understand the potential risk reduction of switching to pMRTP compared with continuous 3R4F exposure and cessation. The present manuscript describes the study design, setup, and implementation, as well as the generation, processing, and quality control analysis of the toxicology and 'omics' datasets that are accessible in public repositories for further analyses.

C57BL6-pMRTP-SW Study - Explore and Visualize Endpoints in Tableau.

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This workbook allows you to visualize endpoints from the C57BL6 pMRTP Switching inhalation study, from exposure markers to histopathology, bronchoalveolar lavage fluid characterization, to transcriptomics and proteomics.

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