8-month systems toxicology inhalation / cessation study with THS2.2 in Apoe‑/‑ mice

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Smoking cigarettes is a major risk factor in the development and progression of cardiovascular disease (CVD) and pulmonary disease (e.g., COPD). That is why modified risk tobacco products (MRTPs), i.e. products with the potential to reduce individual risk and population harm in comparison to smoking cigarettes, are developed.
PMI's MRTPs are in various stages of development and commercialization, and PMI is conducting extensive and rigorous scientific studies to determine whether claims for such products of reduced exposure to harmful and potentially harmful constituents (HPHCs) in smoke are supported.

Utilizing a switching study concept allows to compare the degree of similarity between (i) switching to a candidate MRTP, the tobacco heating system 2.2 (THS2.2) 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


The study reported here aimed at investigating hallmarks of COPD and CVD over an 8-month period in apolipoprotein E-deficient (Apoe-/-) mice exposed to smoke from the 3R4F reference cigarette (CS), to the aerosol of a candidate MRTP, Tobacco Heating System (THS) 2.2., to cessation, or switching to THS2.2 after 2 months of initial CS exposure.

At designated time points (months 1, 2, 3, 6, and 8 after commencing exposure), the animals were examined for multiple parameters to comprehensively assess the development and progression of COPD or CVD indicators. These included hematology and clinical chemistry, pulmonary inflammation measurements, assessment of pulmonary function, lung histopathology and morphometry, evaluation of atherosclerotic plaque formation in the aortic arch and descending aorta, and the examination of molecular changes in various tissues throughout the exposure period by transcriptomics, proteomics, and lipidomics.

Study Design and Conduct

Study design

The study design included five groups of female Apoe-/- mice:

  1. Sham (exposed to fresh air)
  2. 3R4F (exposed to mainstream smoke from the reference cigarette 3R4F, 600 mg total particulate matter (TPM)/m3 equivalent to 29.9 µg nicotine/l)
  3. THS2.2 (exposed to mainstream aerosol from a candidate MRTP: the Tobacco Heating System version 2.2, in which the nicotine level was matched to that of 3R4F)
  4. Cessation of exposure
  5. Switch (to THS2.2)

Choice of switching time point

The overall study design and time point for switching (after 2 months of exposure to 3R4F) were chosen based on the results of previously conducted inhalation studies on C57BL/6 and Apoe-/- mice indicating that lung inflammation, emphysema, and changed pulmonary function were apparent after 2 months of exposure to 3R4F1-3 and another study that showed that Apoe-/- mice readily develop plaques after 1 month of exposure to cigarette smoke4.

Choice of gender

Only female mice were chosen because of a possible increased susceptibility to develop emphysema5.

Choice of endpoints

This integrative study design involved a combined assessment of cardiovascular and respiratory disease mechanisms using classical toxicological end points (including histopathology, physiological measurements, and biomarkers) that were mechanistically substantiated with additional transcriptomics, proteomics, and lipidomics investigations in one study.

A systems toxicology study

This approach was effective in minimizing the number of experimental animals: compared to separate studies for CVD and COPD, and for classical as well as for molecular toxicology, the extra animals that would be required for bridging between studies, the confirmation of biological outcomes, and positive / negative controls, can be saved.

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.

ApoE mice 8 months (THS2.2) Results

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Publications and Reports

Phillips, B. et al. Toxicological sciences 149: 411-432 (2016)

An 8-Month Systems Toxicology Inhalation/Cessation Study in Apoe-/- Mice to Investigate Cardiovascular and Respiratory Exposure Effects of a Candidate Modified Risk Tobacco Product, THS 2.2, Compared With Conventional Cigarettes.

Smoking cigarettes is a major risk factor in the development and progression of cardiovascular disease (CVD) and chronic obstructive pulmonary disease (COPD). Modified risk tobacco products (MRTPs) are being developed to reduce smoking-related health risks. The goal of this study was to investigate hallmarks of COPD and CVD over an 8-month period in apolipoprotein E-deficient mice exposed to conventional cigarette smoke (CS) or to the aerosol of a candidate MRTP, THS2.2. In addition to chronic exposure, cessation or switching to THS2.2 after 2 months of CS exposure was assessed. Engaging a systems toxicology approach, exposure effects were investigated using physiology and histology combined with transcriptomics, lipidomics, and proteomics. CS induced nasal epithelial hyperplasia and metaplasia, lung inflammation, and emphysematous changes (impaired pulmonary function and alveolar damage). Atherogenic effects of CS exposure included altered lipid profiles and aortic plaque formation. Exposure to THS2.2 aerosol (nicotine concentration matched to CS, 29.9 mg/m3) did not induce lung inflammation or emphysema, nor did it consistently change the lipid profile or enhance the plaque area. Cessation or switching to THS2.2 reversed the inflammatory responses and halted progression of initial emphysematous changes and the aortic plaque area. Biological processes, including senescence, inflammation, and proliferation, were significantly impacted by CS, but not by THS2.2 aerosol. Both, cessation and switching to THS2.2 reduced these perturbations to almost sham exposure levels. In conclusion, in this mouse model cessation or switching to THS2.2 retarded the progression of CS-induced atherosclerotic and emphysematous changes, while THS2.2 aerosol alone had minimal adverse effects.

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.

Lo Sasso, G. et al. Journal of translational medicine 14:146 (2016)

The Apoe(-/-) mouse model: a suitable model to study cardiovascular and respiratory diseases in the context of cigarette smoke exposure and harm reduction.

Atherosclerosis-prone apolipoprotein E-deficient (Apoe-/-) mice display poor lipoprotein clearance with subsequent accumulation of cholesterol ester-enriched particles in the blood, which promote the development of atherosclerotic plaques. Therefore, the Apoe-/- mouse model is well established for the study of human atherosclerosis. The systemic proinflammatory status of Apoe-/- mice also makes them good candidates for studying chronic obstructive pulmonary disease, characterized by pulmonary inflammation, airway obstruction, and emphysema, and which shares several risk factors with cardiovascular diseases, including smoking. Herein, we review the results from published studies using Apoe-/- mice, with a particular focus on work conducted in the context of cigarette smoke inhalation studies. The findings from these studies highlight the suitability of this animal model for researching the effects of cigarette smoking on atherosclerosis and emphysema.

Lo Sasso, G. et al. Inhalation toxicology, 28(5):226-240 (2016)

Effects of cigarette smoke, cessation and switching to a candidate modified risk tobacco product on the liver in Apoe-/- mice – a systems toxicology analysis.

The liver is one of the most important organs involved in elimination of xenobiotic and potentially toxic substances. Cigarette smoke (CS) contains more than 7000 chemicals, including those that exert biological effects and cause smoking-related diseases. Though CS is not directly hepatotoxic, a growing body of evidence suggests that it may exacerbate pre-existing chronic liver disease. In this study, we integrated toxicological endpoints with molecular measurements and computational analyses to investigate effects of exposures on the livers of Apoe-/- mice. Mice were exposed to 3R4F reference CS, to an aerosol from the Tobacco Heating System (THS) 2.2, a candidate modified risk tobacco product (MRTP) or to filtered air (Sham) for up to 8 months. THS2.2 takes advantage of a "heat-not-burn" technology that, by heating tobacco, avoids pyrogenesis and pyrosynthesis. After CS exposure for 2 months, some groups were either switched to the MRTP or filtered air. While no group showed clear signs of hepatotoxicity, integrative analysis of proteomics and transcriptomics data showed a CS-dependent impairment of specific biological networks. These networks included lipid and xenobiotic metabolism and iron homeostasis that likely contributed synergistically to exacerbating oxidative stress. In contrast, most proteomic and transcriptomic changes were lower in mice exposed to THS2.2 and in the cessation and switching groups compared to the CS group. Our findings elucidate the complex biological responses of the liver to CS exposure. Furthermore, they provide evidence that THS2.2 aerosol has reduced biological effects, as compared with CS, on the livers of Apoe-/-mice.

Szostak, J. et al. Food and Chemical Toxicology, 101:157-167 (2017)

Aerosol from Tobacco Heating System 2.2 has reduced impact on mouse heart gene expression compared with cigarette smoke

Experimental studies clearly demonstrate a causal effect of cigarette smoking on cardiovascular disease. To reduce the individual risk and population harm caused by smoking, alternative products to cigarettes are being developed. We recently reported on an apolipoprotein E-deficient (Apoe/-) mouse inhalation study that compared the effects of exposure to aerosol from a candidate modified risk tobacco product, Tobacco Heating System 2.2 (THS2.2), and smoke from the reference cigarette (3R4F) on pulmonary and vascular biology. Here, we applied a transcriptomics approach to evaluate the impact of the exposure to 3R4F smoke and THS2.2 aerosol on heart tissues from the same cohort of mice. The systems response profiles demonstrated that 3R4F smoke exposure led to time-dependent transcriptomics changes (False Discovery Rate (FDR) < 0.05; 44 differentially expressed genes at 3-months; 491 at 8-months). Analysis of differentially expressed genes in the heart tissue indicated that 3R4F exposure induced the downregulation of genes involved in cytoskeleton organization and the contractile function of the heart, notably genes that encode beta actin (Actb), actinin alpha 4 (Actn4), and filamin C (Flnc). This was accompanied by the downregulation of genes related to the inflammatory response. None of these effects were observed in the group exposed to THS2.2 aerosol.

Tableau workbook - Visualize Data from the Apoe-/- THS2.2 8-Month Inhalation Study (except omics datasets)

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This workbook allows you to visualize endpoints from the apoe-/- Switching inhalation study, from exposure markers to histopathology, bronchoalveolar lavage fluid characterization, lung function...

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