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Cytotoxic Effects of Electronic Cigare3es on 16HBE Human Bronchial Epithelial Cells In Vitro
INTRODUCTION Promoted as a means of reducing smoking, Electronic cigare5es (ECs) have been the subject of much interest contribu=ng to its open considera=on as a safer smoking alterna=ve. Recently approved by the Britain’s medicine regulator for this purpose, sales are expected to grow significantly in the next few years, where prescrip=on through the NHS could become readily available1. However, healthcare prac==oners, remain uncertain of the safety and efficacy of electronic cigare5es as a consequence of limited evidence, inconsistencies in results, methodologies and absence of long-‐term con=nuous studies. Besides chemical evalua=ons2, limited studies have performed in vitro on the airway epithelial; therefore no definite conclusions can be drawn on the poten=al cytotoxic effects and safety of ECs. Thus, in order to compare cellular reac=ons induced of E.liquid and it’s aerosol, the current project aimed to implement a realis=c simula=on of E.C use. We developed an in vitro cytotoxicity model, analyzing a high nico=nic content (18mg/ml) ice mint flavor, Bri=sh e-‐liquid, in order to evaluate the cytotoxic poten=al, with and without pH adjustments, in addi=on to cellular levels of poten=al pro-‐inflammatory cytokine release IL-‐6 and TER of airway epithelial cells 16HBE.
RESULTS
Figure 1: DisrupGon of 16HBE cell lines following exposure to different treatments, (Vape and E.liquid at 1.25% v/v and a control) for different exposure duraGons (4 and 26 hours). 16HBE cells on inserts were challenged apically with E.C and Vape at 1.25 % v/v concentra?ons. An untreated control was also analyzed. TER (Ω cm2) was measured before cell treatment (t=0) and at 4h and 26h respec?vely. Data calculated as a % mean change from pre-‐treatment reading ±SD, 4 replicates, 3 repeats. *represents significant difference in measured TER with respect to the control group; p< 0.05; 2 way ANOVA Tukey.
Figure 2: Change in expression release of IL-‐6 by 16HBE in response to 24 hour exposure to “Vape” and E.liquid (0.306%-‐ 5% v/v) or posiGve control. Il-‐6 release was assed using Human Il-‐6 Elisa set. Absorbance was measured at 450nm, represented as mean values to respec?ve treatments ±SD of 4 replicates. IL-‐6 expression was significantly different for vape and E.liquid (p<0.001); and E.liquid from control (p<0.05), remarkably at 5% v/v for E.liquid (p<0.05) represented by *; Kruskal-‐wallis and post hoc Mann-‐Whitney.
Figure 3: Cytotoxic screening following exposure of a) “Vape” and E.liquid on 16HBE, b) pH treatment and their respecGve controls a) Cytotoxicity, measured from LDH ac?vity of 16HBE aber 24 hours of exposure to treatments at 0.306-‐5% v/v concentra?ons.
Data is presented as mean values ±SD of 10 replicates for each treatment, 18 controls. b) Cytotoxic assessed from LDH ac?vity post 24 hour exposure to pH treatment adjusted to 7.3 from 8.23 (Vape) 8.53 (E.liquid)
revealing strong alkaline proper=es, physiologically incompa=ble with cellular environment and func=on. . Experiments were conducted in 4 replicates; error ±SD (Standard Devia?on).
13815280/MENDES
Disrup.on of Epithelial Barrier Func.on
a) b)
CONCLUSIONS à Cellular events occurring post treatment of
E.liquid and Vape include increase in cytotoxicity and =ght junc=on degrada=on in a dose/=me rela=onship respec=vely (Figure 1 and 3)
à The release of IL-‐6 is independent of dose,
and further suppressed at 5%, presumably due to mass cell death. (Figure 2)
à Cellular cytotoxicity is found to be
sta=s=cally higher in E.liquid compared to Vape, where 4 readings out of 10 were above moderate range cytotoxicity (70%) according to -‐ISO 10993-‐5 protocol.3
à There is a significant effect of pH contribu=ng
towards the cytotoxicity of our cell model.
à These finding are in agreement to several studies, however pH unrecognized issue must be further exploited in order to d e t e rm i n e t h e p o t e n = a l h e a l t h consequences in a long-‐term E. cigare5e use.
à The study proves that E.liquid and vape have
a poten=al to alter the Airway Epithelial morphology, func=on and cell viability, even at low exposure strengths, which are possibly observed concentra=ons of vapor absorbed into the lungs.
MATERIALS AND METHODS Materials: Ice Mint flavor with full strength nico=ne levels 18mg, and a VG/PG ra=o of 65:35 (Liqualites,Bolton,UK) was opted for this experiment. For the produc=on of extracts, a commercially available 160W temperature control device (SMOK x box cube II, SMOK Tech, Shenzhen, China) was used, consis=ng of lithium ba5ery, a triple coil Ni200 alloy, TFV4 atomizer. (SMOK Tech) Known exact % concentra=on of E.liquid vape condensate used. Cell culture and Treatment preparaGon: 16HBE cells were cultured with MEM supplemented with 10% FBS (PAA Laboratories) . Stock solu=ons for E.liquid and Vape were prepared, from which serial dilu=ons were conducted. (5%-‐0.036 %v/v). For cytotoxicity and cytokine experiments, cell were seeded in 96 and 48 well plates respec=vely in 100μL MEM +10% FBS. For transepithelium resistance, cells were seeded into 12 transwell inserts (Corning Incorporated, NY,USA) with DMEM , Hams F-‐12 mix (1:1) (GE HealthcarePAA Laboratories, Austria). pH stocks were adjusted to pH 7.3 from 8.23 (Vape) 8.53 (E.liquid). Transepithelial resistance (TER): Prior seeding 16HBE cells into the 12 transwell inserts, 200μl of collagen was added onto each insert coa=ng (Pure col). 16HBE cells, were then seeded into the apical chamber at a seeding density of 4.3 x 105 cells/well in 500μl of appropriate cell culture medium and further 1500μl of cell culture medium was added to basolateral chamber. Aner 24 hours cells were subjected to air-‐liquid interface, and used on the 7th day following seeding where TER measurements were conducted using Epithelial Tissue Voltohmeter (EVOM) and hand-‐held chops=ck-‐type electrode prior exposure of each variable (E.liquid, Vaped 1.25%v/v and control) and at 4 and 26 hours post treatment respec=vely. Cytokine IL-‐6: Cells were seeded with a density of 1.5 x 104 cells/well and treated with Vape, E.lqiuid or posi=ve control (vanadyl sulphate) in appropriate media for 24 hours under standard condi=ons, aner which IL-‐6 release was measured using a commercially available Human IL-‐6 ELISA kit (BD OptEIA™, Biosciences Pharmingen ,USA). LDH Cytotoxicity assay: Cells were seeded with a density of 1.0 x 104 cells/well in 96-‐well microplates ,in appropriate media overnight. Medium was subs=tuted by treatments or len untreated (control) for 24 hours and successively, evaluated using a Pierce LDH Cytotoxicity Assay Kit. (Thermo Scien=fic, Rockford, USA)
The E.C was ac=vated for 2-‐2.5 sec. every 30 secs. for a period of 1 hour. Successively the extracts from the two collec=ons flasks were combined together.
Cytotoxicity is pH dependent
a) 2 way Anova; Kukey Post Hoc b) Kruskal-‐wallis, and Mann-‐Whitney post hoc.
*Represents the significant difference in cytotoxicity with respect to control treatments p<0.05. nRepresents the significant difference between cytotoxicity with respect to Treatment concentra?on 5 %v/v. p<0.05 ✚Represents significant difference between cytotoxicity of E.liquid and Vape p<0.05.
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References 1-‐ Nico=ne without smoke Tobacco harm reduc=on A report by the Tobacco Advisory Group of the Royal College of Physicians (April 2016) h5ps://www.rcplondon.ac.uk/file/3563/download?token=uV0R0Twz (accessed 05.05.16) 2-‐ Famele, M., C. Ferran=, C. Abenavoli, et al. 'The Chemical Components of Electronic Cigare5e Cartridges and Refill Fluids: Review of Analy=cal Methods', Nico?ne & Tobacco Research, vol. 17/no. 3, (2015), pp. 271-‐279. 3-‐ISO 10993:5 Standard. Biological Evalua=on of Medical Devices—Part 5: Tests for in vitro Cytotoxicity, 2009. Available online: h5p://www.iso.org/iso/home/store/catalogue_tc/ catalogue_detail.htm?csnumber=36406 (accessed on 14 March 2016).
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