The lung in the lab
Building on its considerable experience assessing tobacco smoke, British American Tobacco is developing new methods to understand the impact on human tissue of vapor.
By Marina Murphy
The e-cigarette category is still relatively new, and scientists continue to look for new ways of assessing vapor products, not least because of their potential to improve public health by reducing the death and disease associated with smoking. There are a lot of studies being conducted and a lot of data being produced, and as the producer of Vype, British American Tobacco (BAT) is among the many organizations doing these studies. In this article, Marina Murphy, head of scientific media relations for research and development at BAT, discusses the company’s work to understand the impact of smoke and vapor on human lung tissue.
Lung cancer is one of the deadliest diseases associated with smoking. So understanding the impact of smoke on human lung tissue has been the subject of many years of study for us. The results we have attained and the methods we have developed are proving invaluable in our investigations into the potential adverse effects of e-cigarette vapor on human airway tissue compared with cigarette smoke. For example, we have developed a system that combines smoking/vaping robots with an exposure chamber containing human airway tissue. This system allows us to compare the impact of cigarette smoke and e-cigarette vapor on human lung cells in a realistic way, by delivering aerosols to these tissues at puffing profiles seen in smokers and vapers. Indeed, this system mimics the structure, function and exposure of normal airway tissue more closely than ever before and allows us to conduct all sorts of lab-based biological tests to compare smoke and vapor.
Here I will describe this “lung in the lab,” as well as some of the results we have obtained using it and how we have gone about ensuring that these results can be trusted.
Our method
E-cigarette vapor can contain nicotine, humectants, flavorings and thermal degradation products, so it is important to understand its potential impact on biological systems.
We use a combination of an exposure system containing human tissue and smoking/vaping robots. The robots mimic the exposure when humans inhale by drawing on the cigarette or e-cigarette to produce an aerosol and then delivering this aerosol or just air to the tissue.
A number of different tissue types and systems can be grown in the lab and exposed to the aerosols. For example, we can use simple systems consisting of a single-layer lung cell culture or more complex, multicellular, commercially available 3-D human respiratory tissues—MucilAir and EpiAirway are the most common. These tissues comprise human airway cells that have been cultured to form different layers of cells that resemble the lining of the human respiratory tract, orwindpipe.
The tissues are put in small dishes (about the size of a thimble), where they are exposed to aerosol on one side and nourishing media on the other side—much like they are in the human body. The temperature is maintained at 37 degrees Celsius—body temperature—again to ensure that exposure mimics real-life exposure as closely as possible.
This model allows us to conduct all sorts of lab-based biological tests to compare smoke and vapor—from the classical tests looking at toxicity (DNA damage, cell death, etc.) to newer studies that mimic key events in disease processes, including oxidative stress and genetic changes in the tissue following exposure, to develop a specific fingerprint or panel of disease markers.
In most cases to date, we either see no response or a substantially reduced response to vapor from our Vype ePen e-cigarette in comparison with cigarette smoke in these lab-based tests. In some cases, the impact of the Vype e-cigarette vapor is indistinguishable from that of the laboratory air where the study was conducted.
Clearing the fog
These initial results are amazing, but vapor is compositionally very different from smoke—it’s much harder to see, for a start. It’s far less complex, containing substantially fewer components, especially in respect of the toxicants found in smoke. In order to be confident in our own testing, therefore, we needed to prove that e-cigarette aerosol droplets are actually delivered to the test tissue, rather than leaching out of the system or sticking to the tubing.
To do this, we used microbalances, tiny scales placed in the dishes where the cells would be placed, that can weigh minuscule levels (nanograms) of particles that come in contact with cell surface, to determine the amount of aerosol delivered from cigarettes and e-cigarettes in an exposure chamber. Basically, this technology allows us to measure amounts so small that we can accurately assess numbers of particles (deposited mass) per puff and the amount of deposited nicotine on the surface.
The results show, on a puff-by-puff basis and at a common dilution, that the e-cigarette aerosol deposited greater mass than cigarette smoke in both systems. Nicotine delivery was much greater from the cigarette than from the e-cigarette.
“It may seem counterintuitive that the aerosol that delivered the most mass had the least impact, but it’s about what that mass represents,” explains Marianna Gaca, preclinical assessment manager at BAT’s R&D center.
Smoke droplets carry the products of combustion—thousands of chemicals and hundreds of toxicants—whereas e-cigarette aerosol droplets contain the aerosolized form of the main ingredients that make up e-liquids: humectants, water, nicotine and flavoring.
“This means the mass deposited on the cells by the e-cigarette vapor is compositionally very different to that deposited by cigarette smoke,” says Gaca. We published the results in Chemistry Central Journal (DOI: 10.1186/s13065-016-0221-9).
“We are very excited about these results because it means that we can be confident that we are delivering e-cigarette aerosol to cells in our tests and that we can be confident, therefore, in our results,” says Gaca. “These are important findings, as we have shown in our lab-based chemical and biological tests that e-cigarettes have the potential to reduce risk in comparison to cigarettes. The next steps are to evaluate these products in clinical and population-based studies to ensure that lab-based reductions translate to disease-relevant reductions in man, both at an individual and a population basis.”
BAT has already conducted a series of tests that compare the impact of smoke with that of vapor. These tests mimic key events in the development of tobacco-related diseases, such as cardiovascular disease or chronic obstructive pulmonary disease. We have, for example, shown that in contrast to smoke, e-cigarette vapor has substantially reduced responses in lab-based tests representative of cellular oxidative stress and toxicity, DNA damage, mutagenicity and gene expression.
BAT’s Vype ePen test results to date are in line with current available evidence that e-cigarettes are on the order of 95 percent less risky than conventional cigarettes. Public Health England, an executive body of the U.K. Department of Health, published a report saying that the current expert estimate is that using e-cigarettes is around 95 percent safer than smoking cigarettes, although more research is needed. The Royal College of Physicians has said that the public can be reassured that e-cigarettes are much safer then smoking and that they should be widely promoted as an alternative to cigarettes.
