Heat‐not‐burn tobacco (IQOS), oral fibroblasts and keratinocytes: cytotoxicity, morphological analysis, apoptosis and cellular cycle. An in vitro study

Abstract Objectives The aim of this work is to investigate the biological effects of IQOS smoking on human gingival fibroblasts and human keratinocytes analysing cell viability, morphology, migration, apoptosis and cell cycle. Background Electronic cigarettes and tobacco heating systems have been marketed to reduce smoking damages caused by combustion. Methods Human gingival fibroblasts and human keratinocytes viability was determined by a colorimetric assay measuring mitochondrial dehydrogenase activity (MTT assay); after an in vitro exposure of 24 h, cell morphology was analysed with scanning electron microscope and cell migration was tested by Scratch assay, a method to mimic the migration of the cells during wound healing in vivo. Apoptosis and cell cycle were analysed with flow cytometry, and the expression of related genes (p53, Bcl2, p16 and p21) was indagated using real‐time polymerase chain reaction. Results IQOS extracts increased both cell viability (23%‐41% with fibroblasts and 30%‐79% with keratinocytes) and migration. No morphological alterations were observed. IQOS extracts did not induced an increase in cell death, but rose the number of S‐ and G2/M‐phase cells. IQOS extracts also significantly increased p53 expression by fibroblasts (undiluted and 6.25% dilution, 2‐ and 3.6‐fold higher, respectively) and reduced both Bcl2 (about two‐ and fivefold, respectively) and p21 expressions (about twofold with both extracts), while on keratinocytes both undiluted and 6.25% dilution extracts increased Bcl2 expression (about four‐ and threefold higher, respectively) and reduced p53 expression (about two‐ and fivefold, respectively). Conclusion IQOS smoke seemed to induce proliferation as highlighted by a viability assay, and migration and cell cycle analysis. The increased cell proliferation induced by IQOS devices must be carefully investigated for its possible clinical effects on oral cell populations.


| INTRODUC TI ON
Tobacco smoking is one of the most serious public health problems the world has ever faced. Despite over 8 million deaths per year that can be directly attributed to it, there are still about 1.3 billion smokers worldwide. 1 Smoking is the main cause of mortality due to cardiovascular diseases like atherosclerosis and its consequences, 2 respiratory disorders such as chronic obstructive bronchopathy (COPD), 3,4 chronic bronchitis and pulmonary emphysema, 5 bone disorders such as osteoporosis 6,7 as well as cancer, particularly lung, bladder, liver, breast and oral malignancies. 8,9 Consequently, governments worldwide have promoted anti-smoking campaigns and laws. Many devices were developed to induce smoking cessation like, for example, nicotine patches and chewing gums which, as they did not replicate the smoking ritual, often failed in their aim. 10 Despite this, electronic cigarettes and the IQOS cigarette are still widespread today. As an alternative to tobacco smoking, these devices are assumed to be less harmful but their real effects are still not known.
The IQOS battery-powered heated tobacco product (HTP), which is manufactured by Philip Morris International, was launched in 2014 and approved by the US Food and Drug Administration (FDA) for the US market in April 2019. Today it is found on 51 markets in Europe, South America and Asia. 11 Resembling a traditional cigarette, the battery-powered IQOS device contains a tobacco cartridge of about 3 cm in length with or without a filter. The cartridge is inserted into a self-heating cylinder in the portable battery which, thanks to aluminium foil, heats it up to a temperature of about 350℃. 12 The kit also contains a battery charger.
The tobacco cartridges consist of: • Tobacco, treated with glycerine, with 0.5 mg of nicotine • External paper covering thin aluminium foil around the tobacco • A cooling filter in biopolymer • A hollow acetate cellulose mouthpiece around the filter.
The main advantage of the IQOS device lies in its noncombustion of tobacco which is heated to much lower temperature than in traditional cigarettes. In fact, as most of the 0.3 mg-0.8 mg of nicotine and 0.0005 mg of tar burns in a normal cigarette, about 20% is absorbed into the body. 13,14 According to the manufacturers, lack of combustion products and toxic smoke in the IQOS device eliminates or reduces most harmful tobacco-related substances and health risks. 15 Conversely, several studies observed that the IQOS aerosol contained substances derived from pyrolysis and that thermogenic degradation of the tobacco cartridge was similar to traditional cigarette smoke. 15 Toxic components of cigarette smoke such as tar, nicotine, carbonyl compounds (formaldehyde, acrolein, acetaldehyde) and nitrosamines were detected in the IQOS aerosol, thus potentially constituting a risk to human health. 16 Indeed, one analysis showed that the IQOS emissions contained organic acids, nicotine and aldehydes like acetaldehyde, formaldehyde and acrolein, although in lower concentrations than in traditional cigarettes. 17 Some studies even showed that menthol-flavoured IQOS contained even higher concentrations of these harmful components. 18 To date, the very few scientific studies who have investigated IQOS toxicity have mainly focused on the IQOS device itself and on comparing its aerosol composition with traditional and electronic cigarettes. 19 Several controversies have emerged over the quantity of harmful substances that IQOS releases, whether its emission should be classified as an aerosol or smoke and, if smoke, the effects of passive smoking. 20,21 In vitro studies demonstrated that the IQOS aerosol toxicity was intermediate between electronic and traditional cigarettes. Furthermore, it damaged respiratory tract cells, the only cells that have been investigated to date. 22,23 As the oral cavity is the first anatomic structure to come into contact with cigarette smoke and the IQOS aerosol, an analysis seemed required of their effects on specific oral cell populations.
The present in vitro study assessed the biological effects of IQOS cigarette smoking on oral fibroblasts and keratinocytes. Specifically, viability was determined by the MTT assay, morphology by SEM analysis and migration by the scratch wound assay. Apoptosis and cell cycle were analysed by flow cytometry. Expression levels of p53, Bcl2, p16 and p21 genes were assessed by RT-PCR.
The first null hypothesis is that the biological effects caused by IQOS devices are similar to those generated by traditional cigarette.
The second null hypothesis is that the results obtained from this in vitro study do not allow to advance suppositions on damages generated by IQOS device.

| Study design
The aim of this study is to compare the effects of the IQOS aerosol on exposed vs non-exposed human gingival fibroblasts and human keratinocytes.

| Cytotoxicity assay (MTT)
Human gingival fibroblasts and human keratinocytes were seeded

| Morphological analysis (SEM)
To determine the effects of extracts on cell morphology, human gingival fibroblasts and human keratinocytes were grown on a cover glass (

| Scratch assay
To investigate fibroblast and keratinocyte migration, cells were plated on 6-well flat bottom microtitre plates (Thermo Fisher Scientific) and grown in 2 mL medium. At 90% confluence, medium was removed and, using a sterile P-200 pipette tip, a straight scratch was done along the monolayer in the well centre, as described elsewhere. 29

| RNA isolation and RT-PCR analysis
To analyse apoptosis and cell cycle genes, human gingival fibroblasts and keratinocytes were seeded (1 × 10 5 cells/ml) in 6-well flat bottom microtitre plates (Thermo Fisher Scientific). At confluence, cells were treated with 6.25% diluted and undiluted extracts or fresh medium (control groups) for 24 h.
Total RNA was isolated as described elsewhere. 29 Briefly, RNA from control and treated fibroblasts/keratinocytes was isolated using a total RNA purification kit (Thermo Fisher Scientific) and quantified by reading the OD at 260 nm on a BioPhotometer

| Apoptosis and cell cycle analysis
Apoptosis and cell cycle analysis were assessed by flow cytometry as previously described. 29 Briefly, 24 h after treatment with undiluted Data were analysed using FlowJo software (TreeStar).

| Cytotoxicity assay (MTT)
At all dilutions, the IQOS extract increased human gingival fibroblast viability by a mean of 30% (range 23% with undiluted extract -41%
Viability increases in both cell lines were due to a proliferation stimulus. Consequently, the following experiments used only undiluted extract and 6.25% diluted.

| Morphological analysis (SEM)
Besides confirming cell proliferation (Figure 2A), SEM analysis showed undiluted and 6.25% diluted IQOS extracts induced only cell surface corrugation in gingival fibroblasts ( Figure 2B, C) compared with control cells. Neither IQOS extract impacted on strong fibroblasts substrate adherence. As in control cells, several filopodia and lamellipodia were observed, indicating adhesion and migration ( Figure 2B, C).
Compared with controls, keratinocyte proliferation was greater with the 6.25% diluted extract than with the undiluted ( Figure 3A) and more filopodia and lamellipodia were observed ( Figure 3B, C) which were long and fine after exposure to undiluted extract and short and thick after exposure to the 6.25% diluted ( Figure 3D).

| Scratch test
Both IQOS extracts induced a similar, non-significant increase in migration in gingival fibroblasts compared with controls. After 24 h, all wounds were closed ( Figure 4A, B).  Figure 5A, B).

| Apoptosis and cell cycle analyses
Flow cytometry detected no significant difference in fibroblast and keratinocyte apoptosis 24 h after exposure to undiluted and 6.25% diluted IQOS extracts (Figures 6A and 7A). Both extracts significantly increased in the percentage of each cell in the S (p < .001 for both) and G2/M (p < .05 for both) cell cycle phases ( Figures 6B and 7B).

| DISCUSS ION
In the human body smoke first encounters the oral cavity and comes the present study focused on human keratinocytes and human gingival fibroblasts.
The first difficulties we had to overcome were capturing all the aerosol components given their volatility and administering them to cell cultures. We designed a smoke production mechanism consisting of a vacuum pump that sucked the IQOS aerosol into a flask containing culture medium which absorbed all its components. It was needed because, even though cigarette toxicology studies used smoke machines with fixed smoke conditions, exposure protocols had been shown to be sub-optimal because cigarette aerosols were altered. 44,45 Another difficulty lay in testing the same quantity of aerosol quantity as found in IQOS consumers. The Exposure to IQOS extracts did not alter the morpho-functional status in fibroblasts but did increase filopodia and lamellipodia, cytoplasmic extensions in keratinocytes. The keratinocyte response to IQOS exposure might be an attempt to achieve greater stability by maintaining firm contact with the substrate and neighbouring cells.
The IQOS extract did not affect human osteoblast morphology, as assessed by the length of primary cilia which are present during their differentiation. 12 Innovative findings in the present study were that all IQOS concentrations induced a greater cell migration than control, as shown by the scratch test results which, as expected from the MTT results, was more marked in keratinocytes than in fibroblasts, particularly with the 6.25% IQOS extract.
IQOS exposure was associated with lack of apoptosis, a common rates of cytotoxicity and apoptosis. 50 Although many key apoptotic proteins that are activated or inactivated in apoptotic pathways have been identified, the molecular mechanisms underlying activation are not fully understood and are the focus of ongoing research. 51 In an attempt to reconcile these apparently contradictory results, we might speculate that aldehyde, acreoline and heavy metal concentrations in the IQOS aerosol, 41 could damage DNA by increasing strand breaks, triggering an initial mutagenicity/genotoxicity 52 that was not yet detectable in the apoptosis rate. Assessment of tumour and proliferating genes will be needed to confirm this hypothesis. Alternatively, post-transcriptional p53 gene modifications might prevent an increase in cell death due to apoptosis.
In conclusion, the clinical significance of the results of this preliminary investigation into the effects of the IQOS device are hard to discern. Our innovative system indicated that IQOS was not toxic for oral fibroblasts and keratinocytes as it neither modified survival nor morphology. Since it did impact on proliferation and the cell cycle further studies are needed to identify which components of the IQOS aerosol are involved. Another limitation of this study was restricting IQOS exposure to 24 h which precluded long-term observations. So, it might be interesting to study a treatment repeated over time (chronic).
Even if the smoke system used in the study does not generate thermal alterations induced by the IQOS device, a further limitation of the study is represented by the need to evaluate the effects of the heating on the oral mucosa. Starting from these preliminary results our null hypotheses can be rejected.