Scholarly article on topic 'The comparative in vitro assessment of e-cigarette and cigarette smoke aerosols using the γH2AX assay and applied dose measurements'

The comparative in vitro assessment of e-cigarette and cigarette smoke aerosols using the γH2AX assay and applied dose measurements Academic research paper on "Environmental engineering"

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Abstract of research paper on Environmental engineering, author of scientific article — David Thorne, Sophie Larard, Andrew Baxter, Clive Meredith, Marianna Gaҫa

Abstract DNA damage can be caused by a variety of external and internal factors and together with cellular responses, can establish genomic instability through multiple pathways. DNA damage therefore, is considered to play an important role in the aetiology and early stages of carcinogenesis. The DNA-damage inducing potential of tobacco smoke aerosols in vitro has been extensively investigated; however, the ability of e-cigarette aerosols to induce DNA damage has not been extensively investigated. E-cigarette use has grown globally in recent years and the health implications of long term e-cigarette use are still unclear. Therefore, this study has assessed the induction of double-strand DNA damage in vitro using human lung epithelial cells to e-cigarette aerosols from two different product variants (a “cigalike” and a closed “modular” system) and cigarette smoke. A Vitrocell® VC 10 aerosol exposure system was used to generate and dilute cigarette smoke and e-cigarette aerosols, which were delivered to human bronchial epithelial cells (BEAS-2Bs) housed at the air-liquid-interface (ALI) for up to 120min exposure (diluting airflow, 0.25–1L/min). Following exposure, cells were immediately fixed, incubated with primary (0.1% γH2AX antibody in PBS) and secondary antibodies (DyLight™ 549 conjugated goat anti-mouse IgG) containing Hoechst dye DNA staining solution (0.2% secondary antibody and 0.01% Hoechst in PBS), and finally screened using the Cellomics Arrayscan VTI platform. The results from this study demonstrate a clear DNA damage-induced dose response with increasing smoke concentrations up to cytotoxic levels. In contrast, e-cigarette aerosols from two product variants did not induce DNA damage at equivalent to or greater than doses of cigarette smoke aerosol. In this study dosimetry approaches were used to contextualize exposure, define exposure conditions and facilitate comparisons between cigarette smoke and e-cigarette aerosols. Quartz crystal microbalance (QCM) technology and quantified nicotine delivery were both assessed at the exposure interface. Nicotine was eluted from the QCM surface to give a quantifiable measure of exposure to support deposited mass. Dose measured as deposited mass (μg/cm2) and nicotine (ng/mL) demonstrated that in vitro e-cigarette exposures were conducted at doses up to 12–28 fold to that of cigarette smoke and demonstrated a consistent negative finding.

Academic research paper on topic "The comparative in vitro assessment of e-cigarette and cigarette smoke aerosols using the γH2AX assay and applied dose measurements"

Toxicology Letters 265 (2017) 170-178

Contents lists available at ScienceDirect

Toxicology Letters

journal homepage www.elsevier.com/locate/toxlet

The comparative in vitro assessment of e-cigarette and cigarette smoke aerosols using the gH2AX assay and applied dose measurements

David Thorne*, Sophie Larard, Andrew Baxter, Clive Meredith, Marianna Gaga

British American Tobacco, R&D Centre, Southampton, Hampshire, SO15 8TL, UK

CrossMark

HIGHLIGHTS

• Cigarette smoke generates a dose dependent increase in double strand breaks.

• E-cigarette aerosols did not generate any increases in double strand breaks above control levels.

• E-cigarette exposures were approximately 28-fold greater than that of cigarette smoke exposures.

• In vitro dosimetry ensures accurate interpretation of the data, and confirms delivery of test agent.

• In vitro dosimetry allows cross-product, study and system comparisons.

ARTICLE INFO

ABSTRACT

Article history:

Received 3 November 2016

Received in revised form 2 December 2016

Accepted 7 December 2016

Available online 10 December 2016

Keywords:

Dosimetry

Cigarette smoke

E-cigarette

Aerosols

g-H2AX

In vitro

DNA damage can be caused by a variety of external and internal factors and together with cellular responses, can establish genomic instability through multiple pathways. DNA damage therefore, is considered to play an important role in the aetiology and early stages of carcinogenesis. The DNA-damage inducing potential of tobacco smoke aerosols in vitro has been extensively investigated; however, the ability of e-cigarette aerosols to induce DNA damage has not been extensively investigated.

E-cigarette use has grown globally in recent years and the health implications of long term e-cigarette use are still unclear. Therefore, this study has assessed the induction of double-strand DNA damage in vitro using human lung epithelial cells to e-cigarette aerosols from two different product variants (a "cigalike" and a closed "modular" system) and cigarette smoke. A Vitrocell® VC 10 aerosol exposure system was used to generate and dilute cigarette smoke and e-cigarette aerosols, which were delivered to human bronchial epithelial cells (BEAS-2Bs) housed at the air-liquid-interface (ALI) for up to 120 min exposure (diluting airflow, 0.25-1 L/min). Following exposure, cells were immediately fixed, incubated with primary (0.1% gH2AX antibody in PBS) and secondary antibodies (DyLight™ 549 conjugated goat anti-mouse IgG) containing Hoechst dye DNA staining solution (0.2% secondary antibody and 0.01% Hoechst in PBS), and finally screened using the Cellomics Arrayscan VTI platform.

The results from this study demonstrate a clear DNA damage-induced dose response with increasing smoke concentrations up to cytotoxic levels. In contrast, e-cigarette aerosols from two product variants did not induce DNA damage at equivalent to or greater than doses of cigarette smoke aerosol. In this study dosimetry approaches were used to contextualize exposure, define exposure conditions and facilitate comparisons between cigarette smoke and e-cigarette aerosols. Quartz crystal microbalance (QCM) technology and quantified nicotine delivery were both assessed at the exposure interface. Nicotine was eluted from the QCM surface to give a quantifiable measure of exposure to support deposited mass. Dose measured as deposited mass (mg/cm2) and nicotine (ng/mL) demonstrated that in vitro e-cigarette exposures were conducted at doses up to 12-28 fold to that of cigarette smoke and demonstrated a consistent negative finding.

© 2016 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-

ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Abbreviations: ACM, aerosol collected mass; ALI, air liquid interface; ATCC, American type culture collection; BEAS-2Bs, human bronchial epithelial cells; BEGM, bronchial epithelial cell growth medium; CRM No 81, CORESTA Recommended Method No 81; E-cigarettes, electronic cigarettes; GC, gas chromatography; HCI, health Canada intense puffing regimen; HPLC, high performance liquid chromatography; ISO, International Organisation of Standardisation; PG, propylene glycol; QCM, quartz crystal microbalance; RCC, relative cell counts; TPM, total particulate matter; UPLC-MS/MS, ultra-performance liquid chromatography tandem mass-spectrometry; VG, vegetable glycerol.

* Corresponding author. E-mail address: David_Thorne@bat.com (D. Thorne).

http://dx.doi.org/10.1016/j.toxlet.2016.12.006

0378-4274/© 2016 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Non-clinical in vitro studies have been widely established and utilised to support the toxicological evaluation of chemicals and complex mixtures including cigarette smoke. In particular, in vitro studies have been utilised to assess the genotoxicity, mutagenicity and cellular response of tobacco smoke particulate matter (Andreoli et al., 2003; Johnson et al., 2009). The detection of gH2AX is an emerging in vitro option for the analysis of DNA damage/repair. It works on the premise of the phosphorylation of H2AX, a histone nuclear protein, in response to specifically double strand breaks (Rogakou et al., 1998). In recent years the assessment of in vitro H2AX phosphorylation (forming gH2AX) has been extensively investigated and employed for the assessment of DNA damage in a variety of scenarios and fields of research (Dickey et al., 2009; Garcia-Canton et al., 2013). It has further been suggested that this endpoint could compliment the current battery of genotoxicity tests for the assessment of tobacco smoke (Garcia-Canton et al., 2012). A key advantage of the gH2AX over other DNA strand break endpoint assays is its compatibility with high content screening (HCS) techniques for higher throughput and multiplexing potential. Measurement of H2AX phosphorylation has been employed for the assessment of tobacco smoke predominately using submerged exposure scenarios. However, advances in aerosol generation and delivery technologies has given rise to in vitro aerosol exposure systems capable of delivering aerosols to an air-liquid exposure interface (ALI). This technology has offered opportunities to enable more advanced screening techniques and sophisticated exposure scenarios, using 2D and 3D cell systems cultured at the ALI (Klus et al., 2016).

Currently there are a variety of different bespoke and commercially available options for in vitro tobacco aerosol exposure (Thorne and Adamson, 2013). Irrespective of set up, all in vitro aerosol exposure systems utilise the same principles. Aerosol is generated, normally via a syringe/piston, the puff of aerosol is then diluted using a variety of dilution principles specific to the setup, and delivered to a cell system normally housed at the ALI in specific discrete chambers under sterile conditions (Phillips et al., 2005). The development of in vitro aerosol exposure systems have undoubtedly advanced the use and application of in vitro methods for tobacco toxicology assessment. It has allowed researchers to generate and deliver an aerosol to a fully differentiated cell system, thus more closely mimicking in vitro the human smoking condition (Maunders et al., 2007). However, these exposure systems do have associated challenges that need considering before their use for tobacco and nicotine aerosol assessment. Current in vitro aerosol ALI exposure systems utilise many different ways to control dose. The dose within these systems is normally defined by the dilution principle. For example, dose can be managed through a time course (mins), it can be adjusted using airflow (L/min), presented as a ratio of aerosol in air (v:v), or even based on product puff number (/puff) or as a percentage of total delivered smoke (%) (Thorne and Adamson, 2013). The management of dose through dilution principles within these exposure systems often leaves the resulting data difficult to interpret and almost impossible to compare between laboratories and exposure setups and gives little indication of actual delivered dose. Many ofthese methods for managing dose and expressing data are arbitrary and do not facilitate cross-study or platform comparisons. Furthermore, arbitrary dilution principles do not mimic or translate to human exposure.

Presently, none of the ways of expressing data based on dilution principle offer an indication of cellular exposure. Quantifying dose at the cellular level, or exposure interface

through in vitro dosimetry approaches is a fast emerging and growing field of research. In vitro dosimetry approaches have been used to characterise the exposure setup, as a quality control (QC) measure for exposure, and recently used in an inter-laboratory study to compare exposure systems (Majeed et al., 2014; Adamson et al., 2014; Thorne et al., 2015). Dosimetry techniques could be used to compare data across contrasting systems and laboratories and may offer a means to align dose across studies. This is of particular importance, if dose can be aligned across studies and laboratories irrespective of exposure system and experimental setup, then in vitro dosimetry could provide a means to bridge the gaps between diverse studies and may offer a viable link between human, in vivo and in vitro comparisons. This becomes particularly important when comparing the toxicological impact of cigarette use to novel tobacco or nicotine products such as electronic cigarettes (e-cigarettes).

Global e-cigarette use has grown significantly over the last few years (Ayers et al., 2011; Etter et al., 2011) and potentially offers a safer alternative to conventional tobacco products (Biener and Hargreaves, 2015; Ay and Kacker, 2014; Hajek et al., 2014). The current understanding from the available literature suggests that e-cigarettes are significantly less harmful compared to traditional cigarette smoke as assessed under a variety of conditions and test matrices (McNeill et al., 2015; RCP, 2016; Farsalinos and Polosa, 2014; Romagna et al., 2013; Bahl et al., 2012; Farsalinos et al., 2013; Cervellati et al., 2014; Misra et al., 2014; Scheffler et al., 2015). However, a comprehensive understanding of the health risks associated with e-cigarette use is difficult, due to the range of devices, continuing category evolution, variability in consumer behaviour and the absence of regulated manufacturing standards (Farsalinos and Polosa, 2014).

As the health debate around e-cigarettes continues and with the array of e-cigarette options available to the consumer, it has never been more important to investigate the health implications across this new and evolving category. In contrast to cigarette smoke, which has been extensively investigated, e-cigarette aerosols remain relatively poorly understood and characterised in vitro. Due to the level of innovation and customisation, there are a vast array of different e-cigarette permutations available to the consumer. A simple e-cigarette or "cigalike" product comprises of a battery, microprocessor, and an e-liquid. The e-liquid is delivered to a coil that is heated upon activation to create an aerosol stream. E-cigarettes can be activated via puffing which triggers coil activation, or via a button which pre-heats the coil prior to puffing. Recent advances of modular devices have seen the incorporation of larger, rechargeable batteries for more power, an e-liquid tank that can be refilled through standard or personalised mixtures, coil upgrades and variable and controllable power options, all of which are designed to facilitate an increase in aerosol generation and product performance.

In this paper we have assessed phosphorylation of H2AX using in vitro human lung epithelial cells (BEAS-2B) to assess the DNA damaging potential of e-cigarette aerosols generated using a Vitrocell® VC 10 exposure system. Freshly generated aerosol from two commercially available e-cigarette variants (Vype eStick and ePen) were assessed and compared to smoke from a scientific reference cigarette (3R4F). To assess dosimetry and exposure during the study two exposure markers were used: deposited nicotine (ng/mL) and deposited mass (mg/cm2), measured from the exposure interface. The data from this study is presented not as a standard dilution relative to the exposure setup, but as a function of the measured in vitro dosimetry, to facilitate wider comparisons.

2. Materials and methods

2.1. Chemicals and reagents

All chemicals and reagents were obtained from Sigma-Aldrich (Gillingham, UK) unless otherwise stated.

2.2. Aerosol products

The emissions of three aerosol products were assessed in this study, Kentucky Reference cigarettes (3R4F) and two commercially available e-cigarette formats. 1, a puff activated closed "cigalike" device (eStick); 2, a "closed modular" system, dual voltage, button activated product (ePen).

Kentucky 3R4F reference cigarettes were obtained from the University of Kentucky, USA. Prior to analysis, cigarettes were conditioned at least 48 h at 22 ±1 °C and 60 ± 3 % relative humidity in accordance with the International Organisation of Standardization (ISO) guideline, ISO 3402:1999. Both e-cigarettes were obtained from Nicoventures Trading Ltd., UK (www.govype. com). Vype e-liquid cartridges, were formulated in the UK, using pharmaceutical/food grade ingredients and were stored at room temperature prior to use. A breakdown of the product specifications and aerosol generation parameters used in the study can be found in Table 1.

2.3. Cell culture

Human bronchial epithelial cells (BEAS-2Bs) were obtained from the American Type Culture Collection (ATCC). BEAS-2Bs were maintained at 37 ±1 °C in an atmosphere of 5% CO2 in air in Bronchial Epithelial Cell Growth Medium (BEGM). BEGM consisted of Bronchial Epithelial Basal Medium with a SingleQuots kit containing growth factor, cytokines and other supplements

including: bovine pituitary extract, insulin, retinoic acid, transferrin, triodothyronine, epinephrine, human epidermal growth factor.

For ALI exposure, cells were prepared on 24 mm permeable membranes (Transwells®, Fisher Scientific, UK) by seeding 2.4 x 105 cells in 1 mL BEGM, supplemented by a further 2 mL BEGM, which was added to the well beneath each Transwell®. Cells were incubated for approximately 24 h to achieve ~70% confluent monolayers which were confirmed by microscopic observation.

2.4. Aerosol exposure

A Vitrocell® VC 10 aerosol exposure system (Serial Number— VC10/141209) and stainless steel modules, 6/4 CF (Vitrocell® Systems, Waldkirch, Germany) were used to generate, dilute and deliver aerosols to BEAS-2Bs maintained at the ALI. In brief, the Vitrocell® dilution system uses both airflow (L/min) and vacuum rate (mL/min) to define exposure conditions. Different aerosol concentrations were achieved by increasing or decreasing the diluting airflow. In addition, a vacuum sub-samples aerosols (via negative pressure) from the dilution system into the module. The flow rate of the vacuum dictates the flow rate over the cells and was maintained at 5 mL/min for all treatments. Diluting airflow rates within this system were maintained and monitored using mass flow controllers and mass flow meters (Analyt-MTC GmbH, Mülheim, Germany).

For each experiment, triplicate Transwells® were housed in a Vitrocell® 6/4 CF stainless steel module for exposure to the freshly generated aerosol. In addition, triplicate Transwells® were also housed in a Vitrocell® 6/3 CF stainless steel module for exposure to clean flowing air as a concurrent negative air control (5 mL/min). Trumpet heights within modules were set at 2 mm above the Transwell® membrane/quartz crystal microbalance (QCM) surface. Exposures were conducted to mass (mg/cm2) using QCM

Table 1

Specification of products and aerosol generation parameters.

Characteristics Product

3R4F eStick ePen

Aerosol Reference cigarette E-cigarette Aerosol E-cigarette Aerosol

Cigalike, closed system e- Closed modular e-cigarette system with interchangeable cartridges

cigarette (cartomizers)

Manufacturer University of Kentucky Vype® Vype®

(USA) (Nicoventures, UK) (Nicoventures, UK)

Length (mm) 84 84 153

Diameter (mm) 8 8 20 (10 at mouth piece)

Nicotine content 0.7-2.0 mg/ciga 36mg/mL 18mg/mL

Puff number 8-10a 120-150 250-300

Voltage options? (v) N/A No Yes (3.6 or 4.0)

Voltage used in study (v) N/A 3.7 nominal 4.0

Cartridge usedb N/A Classic Flavourb Blended Tobaccob

Rechargeable? N/A Yes Yes

Count Median Diameter (nm) 160-180a 25 12

Volume Median Diameter 215-230a 252 265

Total Particle Number/Puff 6.7e10-4.1e12a 2.1e11 6.8e12

Exposure Regimen used in HCI CRM No 81 CRM No 81

Puff volume (mL) 55 55 55

Puff frequency (secs) 30 30 30

Puff duration 2 3 3

(secs)

Puff profile Bell Square Square

Vent blocking 100% none none

Pre-coil activation N/A None 1s

Activation N/A Puff Button (hand activated)

a = Dependent on smoking regimen used (ISO vs. HCI).

b =as stated on the pack; HCI = Health Canada Intense regimen; CRM No 81 = CORESTA Recommended Method No 81.

Fig. 1. Cross section of the Vitrocell® VC 10 Smoking Robot and 6/4 CF Stainless mammalian exposure module with a quartz crystal microbalance installed into position 4. A button activated e-cigarette (ePen) is shown but the cigarette (3R4F) and the puff activated e-cigarette (eStick) were puffed in the same manner. Aerosol was drawn into the syringe (ii) and delivered to the dilution bar where diluting air was added (iii). Diluted aerosol was drawn into the module (iv) and deposited on the QCM via negative pressure (v) [adapted from Adamson et al., 2016].

technology at airflows of (0.25-1 L/min), for a maximum of 2 h; nicotine was quantified from the QCM surface following exposure

(Fig. 1).

Reference cigarettes were smoked as per the standard operation of the VC 10. The e-cigarette, eStick was hand loaded into the smoking head and puff activated. Vype ePen was connected directly to the syringe, bypassing the rotary head, using a labyrinth seal. This e-cigarette required button activation and was too large in size to sit within the smoking carousel. Button activation was synchronised with VC 10 and was hand activated 1 s prior to puffing and during the 3 s puff interval.

2.5. Measurement of deposited particulate mass

To define exposure, one QCM (Vitrocell® Systems GmbH, Waldkirch, Germany) was installed into the last position of the 6/4 CF Stainless Steel exposure module. QCM technology has been previously described as a quality tool for assessment of aerosol exposure (Majeed et al., 2014; Adamson et al., 2014; Thorne et al., 2015).

2.6. Quantification of deposited nicotine

Nicotine was quantified using an ultra-performance liquid chromatography tandem mass-spectrometry core system (Waters, UK) and triple quad mass spectrometer (Sciex, UK) (UPLC-MS/MS). Techniques used were based on methods published by Jin et al., 2012 and Onoue et al., 2011. Exposed QCM crystals were removed from their housing units and placed in individual flasks containing 3 mL high performance liquid chromatography (HPLC) grade methanol. 30 mL nicotine-d4 internal standard was added to each flask and shaken for 30 min at 160 rpm to wash the surface deposited nicotine from the crystal surface. A 1 mL aliquot of the extracted nicotine was pipetted into Eppendorfs and condensed in an Eppendorf Concentrator 5301 (Eppendorf, UK) for 80 min at 30 °C. Extracts were re-suspended in 1 mL of 5% acetonitrile in water (v/v) and transferred to GC vials and analysed.

2.7. Assessment of double strand Breaks

The gH2AX assay was based on the Thermo-Scientific validated protocol with slight modifications (ThermoScientific, USA), as described in Garcia-Canton et al., 2013. Etoposide was used for

positive control treatments and was diluted in dimethyl sulfoxide (DMSO) to give an initial 100 mM stock solution. Etoposide stock solution was prepared in medium to a 1 mM concentration prior to dosing. BEAS-2Bs were seeded onto 24 mm Transwells® at 2.4 x 105 to achieve 60-70% confluent monolayers. Positive control treatments were applied to both the apical and basal surface for a maximum of 60 min. For aerosol exposures, BEAS-2Bs were prepared in the same way as described above and Transwells® were housed at the ALI in a Vitrocell® exposure module at room temperature (RT), in contact with basal media, for the duration of exposure (maximum of 3 h). Following exposure, cells were immediately fixed, incubated with 50 mL primary (0.1% gH2AX antibody in PBS) and 50 mL secondary antibodies (DyLight™ 549 conjugated goat anti-mouse IgG) containing Hoechst dye DNA staining solution (0.2% secondary antibody and 0.01% Hoechst in PBS), and finally screened using the Cellomics Arrayscan VTI platform.

Images from the Transwells® were taken using the Cellomics Arrayscan VTI platform analysed with the Target Activation Bioapplication software V.6.6.1.4. The bioapplication protocol was set to count a minimum of 1000 objects (cells) perTranswell®. Two different nuclear stains were measured. Nuclear DNA staining with Hoechst dye was assessed in channel 1 to identify viable cell nuclei. These nuclei then served as the target areas for second channel measurement. Channel 2 measured the phosphorylated form of the histone 2AX (gH2AX), whose fluorescence intensity is directly proportional to the number of double strand breaks (Rogakou et al., 1998).

2.8. Assessment of cell viability

Image acquisition was performed as described above. Nuclear DNA staining (Hoechst dye) was used to identify viable cell nuclei with approximately 2000 cells counted per treatment condition. Viable cell counts from negative controls were defined as 100%. Counts in the treated samples were then compared to the negative control, and the percentage cell viability was calculated and referred to as Relative Cell Counts (RCC). A genotoxic response was only considered positive, if the result met the following criteria; >1.5-fold increase above control level, with RCC>25% (Garcia-Canton et al., 2014). In this study, treatments <25% RCC were excluded from analysis, due to potential cytotoxic interfer-

Table 2

Summary of results.

Product Average delivered Average delivered Average gH2AX RCC Cytotoxic gH2AX Statistical

deposition±SD (mg/cm2) nicotine ± SD (ng/mL) intensity ±SD (IU) (%) classification response1 analysis2

Tobacco 3R4F3 0 0 44.7 ±6.4 >25 -ve - ve

3.1 ±0.3 155.0 ±43.6 101.4 ± 10.9 >25 -ve +ve *

5.4 ±1.8 339.0 ±83.2 118.0 ±21.9 >25 -ve +ve *

10.5 ±1.2 930.3 ±288.9 136.9 ±12.9 >25 -ve +ve *

26.9 ±2.1 2757.5 ±561.9 196.9 ±8.2 <25 +ve N/A N/A

E- eStick4 0 0 44.3 ±4.8 >25 - ve - ve N/S

cigarette 35.2 ±0.9 71.3 ±2.0 657.5 ± 70.4 1653.3 ± 150.1 47.8 ±0.6 53.5 ± 5.3

ePen4 0 42.5 ± 4.1 85.7 ± 2.7 0 700.3 ±94.2 2045 ±327.5 44.5 ±3.2 44.8 ± 1.8 47.7 ±2.0 >25 - ve - ve N/S

1 = Response deemed positive if a 1.5-fold increase was observed over controls, with a >25% RCC.

2 = Statistical analysis conducted using a one-way analysis of variance (95% confidence interval).

3 = Cigarette smoke aerosol assessed up to 120 min using 1 L/min and a 5 mL/min vacuum.

4 = E-Cigarette aerosols assessed up to 120 min using 0.25 L/min and a 5 mL/min vacuum.

IU = Intensity units; RCC = Relative Cell Counts; N/S = no statistical difference; * = statistically different from control above the 1.5 threshold (>p = 0.05); N/A=not assessed due to high levels of cytotoxicity; SD = Standard Deviation; +ve = positive response (a genotoxic response was only considered positive, if the result met the following criteria; > 1.5-fold increase above control level, with RCC >25%); —ve = negative response (treatments < 25% RCC were excluded from analysis, due to potential cytotoxic interference).

2.9. Assay controls

3. Results

The following assay controls were assessed to ensure controlled assay conditions. 1 mM etoposide was used as a positive control. BEAS-2Bs were also assessed under submerged culture conditions, maintained in the incubator, termed incubator controls (Inc). These controls acted as a baseline for optimal cell culture conditions. An air control (Air) was also assessed. In this case BEAS-2Bs were raised to the ALI and maintained in the incubator under normal incubator conditions. Finally, a flowing air (ALI) control was also assessed. These controls assessed the effect of flowing air over the BEAS-2Bs and simulated exposure conditions (5 mL/min).

2.10. Data presentation and statistics

Graphs were generated using Microsoft Excel 2010. Statistical analysis was conducted using Minitab® version 16.1.0 and one-way analysis of variance (ANOVA) to 95% confidence. All assessments were conducted on at least three independent experimental occasions, with three replicates per occasion. Data is presented as mean experimental values +/— experimental standard deviation presented on both x-y axis where applicable.

A summary of all the results obtained, including cytotoxicity can be found in Table 2.

3.1. Assay controls

Assay controls, run concurrently with each exposure demonstrated that there was little effect between incubator (Inc), Airliquid interface (ALI) and flowing air controls (Air). In contrast, the positive control, etoposide, produced a statistically positive response above control background. This data indicates that assay conditions such as raising BEAS-2Bs to the ALI and flowing air conditions of exposure had no effect gH2AX intensity (Fig. 2).

3.2. Assessment of dose

Instead of presenting the data as function of the dilution principle associated with the aerosol generation system, such as%, volume, airflow, a novel approach of assessing delivered exposure markers was employed. Delivered mass (mg/cm2) using QCM technology and nicotine (ng/mL) eluted from the QCM surface was

Fig. 2. Graph showing gH2AX intensity range for controls. Inc=cells maintained in the incubator under submerged conditions; Air = cells raised to the ALI, but maintained in culture conditions within incubator; ALI = cells raised to the ALI and submitted to flowing air conditions of ALI exposure (5 mL/min for duration of exposure); Etoposide = positive control (1 mM 60 min exposure).

used to define exposure conditions. In this study cigarette smoke exposures were conducted up to 26.9 mg/cm2 measured using QCM technology. E-cigarette exposures were conducted up to 85 mg/cm2 as measured using the same QCM technology. Nicotine was eluted from the QCM exposure surface and provided a quantifiable measure of dose to support that of deposited mass. Cigarette smoke exposures were conducted up to 930.3 ng/mL, corresponding with the highest non-cytotoxic dose (10.5 mg/cm2) whereas e-cigarette exposures were conducted up to 2045 ng/mL, corresponding to the highest non-cytotoxic deposited mass dose (85.7 mg/cm2). E-cigarette doses were based on an approximate quadrupling of the highest non-cytotoxic cigarette smoke response (10.5 mg/cm2), the response was then furthered doubled from approximately 40 to 80 mg/cm2. Due to the variable nature of e-cigarette technology and dose matching using a gravimetric weight, this study exposed cells to a 0, 35.2 and 71.3 mg/cm2 of eStick aerosol and 0, 42.5 and 85.7 mg/cm2 of ePen aerosol, which correlate to nicotine readings of 0, 657.5,1653.3 ng/mL nicotine per eStick and 0, 700.3 and 2045 ng/mL nicotine per ePen (Table 2).

3.3. Assessment of cytotoxicity

Cigarette smoke was assessed between a dose range of 026.9 mg/cm2. The highest dose of cigarette smoke (26.9 mg/cm2) was the only dose to elicit a cytotoxic response, as per the established criteria. All other treatments for cigarette smoke (0-

10.5 mg/cm2) and e-cigarette aerosol (0-71 mg/cm2 eStick and 085.7 mg/cm2 ePen) were deemed to be below the cytotoxic threshold. The cigarette smoke dose of 26.9 mg/cm2 was excluded from data analysis and is not presented on any of the graphs.

3.4. Assessment of double strand Breaks

Initial experiments focused on the development of the assay for analysis of cigarette smoke, using 3R4F reference cigarettes. A dose range was identified, providing a positive gH2AX response in the range of 0-26.9 mg/cm2 deposited mass, which equated to an exposure time of approximately 0-60 min. At 26.9 mg/cm2 cell

viabilities (defined by relative cell counts--RCC) were below the

25% threshold and were considered cytotoxic and gH2AX data at this exposure point was not assessed. Doses of 3.1, 5.4 and 10.5 mg/ cm2 demonstrated a significant positive response in observed double strand breaks above control levels and were deemed non-cytotoxic, with RCC above the 25% threshold.

The assessment of e-cigarette aerosols, demonstrated that at 71.3 and 85.7 mg/cm2, eStick and ePen respectively, were non-genotoxic and non-cytotoxic. In comparison, cigarette smoke aerosols were genotoxic at a 3.1 mg/cm2 dose and cytotoxic at 26.9 mg/cm2. In addition to gravimetric mass (mg/cm2) dose measurements, nicotine was also quantified from the QCM exposure interface (ng/mL). Deposited mass gives an assessment of overall exposure, but nicotine represents a quantifiable marker

0 500 1000 1500 2000 2500

Delivered nicotine (ng/mL)

Fig. 3. Graph showing the combined response of cigarette smoke versus e-cigarette aerosol. [A] shows data presented as a function of delivered aerosol mass (mg/cm2). [B] shows data presented as a function of delivered nicotine ng/mL. X-Y Standard Deviation based on gH2AX intensity and dose measurements (mg/cm2 and ng/mL).

of exposure across product categories. Eluting nicotine from the QCM exposure interface demonstrated that cigarette smoke genotoxicity, was observed between nicotine doses of 155930.3 ng/mL nicotine. Conversely, e-cigarette exposures ranged between 0-1653.3 ng/mL nicotine for eStick and 0-2045 ng/mL nicotine for ePen and showed no increase in genotoxicity above background levels (determined by air and incubator controls).

The dosimetry data presented here, demonstrates that exposures were approximately 25 fold higher in the e-cigarette compared to cigarette smoke, (3.1 vs. 85.7 (mg/cm2), when using a gravimetric mass as a measure of dose. At these doses there were no observable responses. Nicotine, shows a similar pattern when used as an exposure marker. When comparing the lowest positive genotoxic dose of cigarette smoke of 155 ng/mL nicotine to eStick and ePen an approximate 12-fold increase in exposure is observed, despite a clear non-genotoxic response. For example, when e-cigarettes (eStick and ePen) were compared to cigarette smoke, a positive response is observed in cigarette smoke at a dose equating to 155 ng/mL nicotine as an exposure marker. E-cigarette exposures for both eStick and ePen were conducted up to 1653.3 ng/mL and 2045 ng/mL respectively, without observing a positive genotoxic response (Fig. 3).

Fig. 4 demonstrates the difference in exposure in eStick and ePen at their highest doses compared to cigarette smoke at the lowest positive genotoxic response (3 mg/cm2 and 155 ng/mL nicotine).

4. Discussion

The objective of this study was to investigate the potential for e-cigarette aerosols to induce DNA damage in vitro, compared to traditional cigarette smoke. Using a Vitrocell® VC 10 exposure system, aerosols were generated and delivered to human lung epithelial cells and the phosphorylation of H2AX was assessed as a marker of double strand DNA damage. Dose was assessed using particulate mass and nicotine deposition. This study has compared two very different e-cigarette devices including a puff activated, rechargeable "cigalike" e-cigarette and a "closed modular" button activated system. Whole aerosol exposure systems are based on dilution principles and the resulting data is normally presented in accordance with the specific dilution principle of the system employed; data may be presented as a diluting airflow (L/min), as an aerosol:air ratio (v/v) or a percentage of aerosol dilution (%). None of these techniques however, give a quantifiable measure of exposure. By using in vitro dosimetry approaches and exposure

markers such as deposited mass (mg/cm2) and quantifying actual delivered nicotine to the exposure interface (ng/mL), the resulting data in this study is expressed as a function of dose rather than an arbitrary dilution principle. This approach facilitates cross-system and cross-study comparisons, where previously comparison of in vitro aerosol data were difficult. Using in vitro dosimetry techniques, this study has defined exposure conditions and demonstrated an 'over-exposure' of e-cigarette aerosols compared to that of cigarette smoke. E-cigarette aerosols for example were dosed approximately 28-fold higher using mg/cm2 and ~12-fold higher using nicotine (ng/mL) as exposure markers. Furthermore, when comparing the relationship between mass and nicotine for 3R4F a ratio of approximately 9.8 mg/cm2:1000 ng/mL is observed. Conversely, eStick and ePen e-cigarette aerosols demonstrated an approximate ratio of mass:nicotine of, 43.2 and 41.1 mg/cm2:1000 ng/mL respectively. This indicates approximately 4 times the amount of mass must be delivered by e-cigarette aerosols, to achieve a comparable nicotine dose to that of 3R4F reference cigarette smoke. In a recent study, Adamson et al., 2016 compared the delivery of 3R4F cigarette smoke and ePen e-cigarette aerosol across multiple exposure systems (Borgwaldt RM20S and Vitrocell VC10) and assessed both nicotine and deposited mass from the exposure interface, comparable to the techniques used in this study. Adamson et al., 2016 demonstrated that across both systems e-cigarette aerosols delivered more mass than cigarette smoke at equivalent nicotine doses, confirming these observations. Based on a per puff analysis of cigarette smoke and e-cigarette aerosols, in vitro data from the Vitrocell® VC10 and Borgwaldt RM20S aerosol exposure systems could be successfully compared despite system differences, demonstrating the importance and application of dosimetric approaches in an in vitro environment.

There is now a growing amount of evidence for the use of in vitro dosimetry approaches with an associated biological endpoint to put dose into perspective. In this study 3 mg/cm2 deposited mass of cigarette smoke aerosol was enough to cause a significant increase in double strand breaks, below the cytotoxic threshold. Kilford et al., 2014 and Thorne et al., 2015; both demonstrated that approximately 3 mg/cm2 deposited mass of cigarette smoke aerosol is enough to cause significant mutations in the Ames assay in a variety of tester strains. Furthermore, Thorne et al., 2013 demonstrated cytotoxic levels were achieved in deposited mass doses exceeding 12 mg/cm2, which is comparable to the range observed in this study. Neilson et al., 2015 pushed e-cigarette exposure out to approximately 120 mg/cm2 deposited mass in a 3D cell system and failed to observe any cytotoxic effects of e-cigarette

Fig. 4. Graph showing the approximate fold difference in exposure for eStick and ePen aerosols compared to 3R4F cigarette smoke using the lowest positive non-cytotoxic response as baseline (3.1 mg/cm2 and 155ng/mL) for exposure markers, deposited mass (mg/cm2) and deposited nicotine (ng/mL).

aerosol compared to air controls. Conversely, using theoretical deposited mass measurements Azzopardi et al., 2016 showed an indication of cytotoxicity in e-cigarette aerosols at theoretical doses exceeding 180 mg/cm2 in a 2D lung cell system.

The biological activity of cigarette smoke has been widely demonstrated in vitro (Andreoli et al., 2003; Johnson et al., 2009) and this data supports those findings. The gH2AX assay has been employed for the assessment of cigarette smoke aerosol on a number of occasions. For example, Garcia-Canton et al., 2014 demonstrated that the gH2AX assay can be combined with another aerosol exposure system, the Borgwaldt RM20S for the assessment of cigarette smoke. The study indicated a low response relationship between dose and genotoxicity. Positive genotoxicity was observed for both 3R4F and M4A cigarette smoke aerosols, however, the response observed was non-dose responsive. Here we have combined the gH2AX assay with an alternative aerosol exposure system and demonstrated a genotoxic dose response relationship.

The data in this study under the experimental conditions failed to elicit any genotoxic response of e-cigarette aerosols above controls levels, which is in direct contrast to the clear genotoxic response generated by cigarette smoke within a relatively short exposure timeframe and dose. These observations are further supported, for example, Thorne et al., 2016 demonstrated that freshly generated ePen e-cigarette aerosol had no mutagenic effect in tester strains TA98 and TA100 following a 3-h exposure, whereas cigarette smoke produced clear mutagenic responses after only 24min. Thorne et al., 2016 further demonstrated no-activity in e-cigarette aerosol collected mass (ACM) generated in a comparable manner to total particulate matter (TPM) in both TA98 and TA100 strains and yet 3R4F cigarette smoke and TPM in the same study was shown to be clearly mutagenic. Taylor et al., 2016 also demonstrated that eStick and ePen captured aerosols induce lower levels of oxidative stress compared to cigarette smoke captured aerosols in an aqueous matrix and 2D lung cell system.

Further studies have demonstrated the chemical burden of e-cigarette aerosol is significantly reduced compared to cigarette smoke. For example, a recent study by Margham et al., 2016 reported the levels of individual chemical emissions from a ePen, were 92-99% lower than those from a 3R4F reference cigarette. Compounds measured included the major e-liquid constituents, nicotine, propylene glycol (PG) and glycerol (VG), recognised impurities in pharmacopoeia quality nicotine and eight species identified as thermal decomposition products of PG or VG (Margham et al., 2016). This observation supports the current understanding that e-cigarette aerosols are relatively simple in comparison to cigarette smoke. Other studies have reported that e-cigarette aerosol emissions are in general, lower than the limit of detection or non-quantifiable for analytes such as, tobacco specific nitrosamines, aromatic amines, CO, polyaromatic hydrocarbons, phenols and metals (Westenberger, 2009; Kim et al., 2011; Tayyarah and Long, 2014; Goniewicz et al., 2014; Margham et al., 2016). Given the low chemical burden of e-cigarette aerosols compared to the complex mixture of cigarette smoke, it is unsurprising that the biological effect of the products assessed in this study were significantly lower than that of cigarette smoke given the available and detectable chemical compounds in the source aerosol. However, the presence of low levels of chemicals does not mean that e-cigarette use is without risk. Levels of carbonyl compounds have been measured up to 9-450 times lower than that observed in cigarette smoke (Goniewicz et al., 2014). Levels can increase significantly when e-cigarettes are "dry-puffed", a phenomenon where the e-liquid is vaporised quicker than it can be replaced, resulting in the e-cigarette coli or wick heating in the absence of e-liquid. The potential presence of carbonyl compounds in e-cigarette aerosols remains a concern and possible source of adverse biological activity. This is a key area for

future studies and one which can be assessed with the use of in vitro exposure systems.

This study has assessed two types of e-cigarette variants (a cigalike (eStick) and closed modular device (ePen)), providing further information on the biological effect of e-cigarette aerosols in vitro. Future studies need to focus on the assessment of other novel modular e-cigarette type devices that may be significantly different in their aerosol delivery, generation and emissions. Finally, the in vitro data presented here is an acute short term aerosol exposure technique. There is a need to investigate more chronic based exposure scenarios to understand and assess the potential impact of e-cigarette use. However, as it stands, this data supports a growing portfolio of data indicating that e-cigarette aerosols are significantly less biologically active compared to that of cigarette smoke.

5. Conclusions

The results demonstrate that cigarette smoke generates a dose dependent increase in genotoxicity via the assessment of double strands breaks using an in vitro human lung epithelial model. The system was pushed to cytotoxic levels, defined by relative cell counts. In contrast, e-cigarette aerosol did not generate any genotoxicity or increases in double strand breaks above that of control levels or produce any measureable decrease in cell viability at doses approximating 28-fold greater than that of cigarette smoke exposure. The conclusions are that, at doses where cigarette smoke is clearly positive for both genotoxicity and cytotoxicity, this study did not detect any increases in genotoxicity for e-cigarette vapour, using an aerosol based in vitro exposure system. Assessment of in vitro dosimetry during cellular exposure enabled an accurate measurement to confirm exposures and define a quantitative approach to delivery. Studies such as this, will become important when confirming the reduction of biological activity of e-cigarette aerosols when compared to cigarette smoke. This study further highlights the importance of applying dosimetric approaches to qualify and quantify exposure. In the context of a negative response as demonstrated here, in vitro dosimetry approaches will be fundamental to facilitate more accurate interpretations of the resulting data, confirm delivery of test agent and enable the data to be presented in a format that appropriately allows cross-product, study and system comparisons across the wider research community.

Declaration of interest

The authors were employees of British American Tobacco at the time the study was conducted. Nicoventures Ltd., UK, is a wholly-owned subsidiary of British American Tobacco.

Authors contributions

David Thorne and Sophie Larard designed and executed all experimental work. Andy Baxter analysed all nicotine samples and David Thorne and Sophie Larard analysed all gH2AX data. David Thorne wrote the manuscript, supported by Sophie Larard. Marianna Gaga and Clive Meredith oversaw all biological testing.

Acknowledgements

The authors would like to thank Carolina Garcia-Canton's technical support and gH2AX training, Benjamin Zainuddin for his help with nicotine extractions, Ross Cabot for his aerosol science support and Annette Dalrymple for her technical contributions. Jason Adamson produced Fig. 1.

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