The primary objective of the study was to evaluate cytotoxicity and genotoxicity of both long- and short-term e-cigarette vapour exposure on a variety of epithelial cells to determine if the vapour is indeed toxic to the cells and to attempt to identify the constituent that causes cell damage.
The VA research team selected three distinct cell lines – normal HaCaT and two from head and neck squamous cell carcinoma (HNSCC) cell lines; UMSCC10B, derived from a metastatic lymph node and HN30, derived from a primary laryngeal tumour – cultured in DMEM at 37 °C and 5% CO2.
The cell lines were then treated with filter-sterilised extract (at 0.2 µm pore-size) from Marlboro Red filter cigarettes (reported as containing 1.2 mg of nicotine per cigarette) and e-cigarette brands V2 and VaporFi. Both e-cigarette brands reported an e-liquid mixture of 70% Propylene Glycol and 30% Vegetable Glycerin at 12mg of nicotine per mL – the researchers also utilised nicotine free variants of the chosen e-liquid.
HaCaT cells were treated for 8 weeks, with the treatment media replaced every three days with 1% e-cigarette extract. However, cells treated with cigarette smoke extract could only be treated for 24 hours due to the high toxicity of the extract. The UMSCC10B and HN30 cell lines were each treated for a week.
Following the treatment of the cell cultures, the researchers utilised comet assay to determine the damage to the cell, based on the length of the comet tail. This method has limited usefulness and is laborious as the tail length increases only at the lowest levels of damage and quickly reaches a maximal point thereby reducing the useful range of the assay.
In addition to comet assay, the researchers utilised immunostaining to specifically identify the biomarkers associated with DNA double-strand breaks and flow cytometry to isolate the distribution of cells across the cell cycle, specifically G1 (cells produce RNA and synthesize protein) and G2 (the cell continues to produce new proteins between synthesis and mitosis). In the UMSCC10B cell line, the researchers identified increased accumulation of arrest in G1, and in HN30 an increase in G2 suggesting that the cell is not able to create the required proteins for mitosis.
To evaluate the extent, and possible mechanisms of cell death (apoptosis) as a result of exposure to e-cigarette vapour the HaCaT cell line was treated for one week with both nicotine containing, and nicotine free extract from the V2 and VaporFi brands – subsequently analysed by flow cytometry and Propidium iodide staining . This approach enabled differentiation between live (as PI does not bond with live cells), necrotic and early/late apoptotic cells.
Apoptosis is programmed cell death and occurs if cells are no longer needed regardless whether the cell is healthy or not and generally exactly balances cell division; as opposed to necrosis where the cells "burst" as a result of acute injury. Apoptosis therefore is not necessarily a "bad thing" as the remaining fragments from the process are absorbed and recycled by neighbouring cells that ingest them. Cells can enter apoptosis as part of normal development, or in response to viral infection, cellular stress, or DNA damage.
The results from this evaluation showed that while cells exposed to nicotine containing extract did show signs of apoptosis, the variations between the two e-cigarette brands (V2 and VaporFi) suggests that nicotine may not in fact be the primary cause as the analysis graphs (Figure 4 in the study) for cells in late apoptosis are remarkably similar. However, necrotic cells were substantially similar across the samples suggesting that the cell damage observed is primarily programmed cell death which occurs regularly in the human body.
The main criticism of this study rests with the method of cell exposure. Contrary to the published conclusion that state at biologically relevant doses the vapourised e-liquids induce increased DNA strand breaks and cell necrosis. However, cells are rarely exposed to vapour continuously to the scale of this study, nor are the cells used in the study by definition like actual human cells.
Looking for effects such as inflammation or DNA strand breaks, cells need to survive exposure to the medium used, in the case of cigarette smoke, the cells died within 24 hours so there was no measurement of effects on DNA strand breaks. The same applies to exposure to e-cigarette vapour.
Due in no small part to the press coverage of this study, Cancer Research UK raised concerns regarding the press covering the topic of e-cigarettes and the research into the technology. The news service behind the original press release issued a correction due to concerns about the inaccurate reporting of the research.
The relative harm compared to smoking is the critical point, that comparison is largely missing as the cigarette exposure only appeared "for comparison" without specific context.
NSP Correspondent - Paul Barnes