Distribution, quantification and toxicity of cinnamaldehyde in electronic cigarette refill fluids and aerosols.

The distribution and concentration of cinnamaldehyde were determined in 39 e-cigarette refill fluids plus 6 duplicates using gas chromatography and mass spectrometry (GC/MS). A cinnamaldehyde toxicity profile was established for embryonic and adult cells using a live cell imaging assay, immunocytochemistry, the comet assay and a recovery assay.

MATERIALS AND METHODS

Refill fluids and authentic standards Refill fluids were purchased at various times from internet vendors, including Freedom Smoke USA (Tucson, Arizona, USA), Global Smoke (Los Angeles, California, USA), Johnson Creek (Johnson Creek, Wisconsin, USA), Red Oak (a subsidiary of Johnson Creek), Tasty Puff (Albuquerque, New Mexico, USA), e-cigexpress (Orlando, Florida, USA), Vaporbomb.com (Barberton, Ohio, USA), Vapormaxx (Richmond, Virginia, USA) and DIY Flavour Shack (Las Vegas, Nevada, USA). 17 18 Refill fluids were stored at 4°C in the dark. Only the sample from Tasty Puff (Sinful Cinnamon) and its duplicate were sold as a do-it-yourself (DIY) product. All others, including the product from DIY Flavour Shack, were sold as refill fluids. Authentic standards were purchased to produce ‘lab-made’ refill fluids. Trans -Cinnamaldehyde was purchased from TCI (Tokyo, Japan), and propylene glycol was from Acros Organics (New Jersey, USA). With exception of the aerosol MTT assays, all toxicity assessment assays were performed at the no observed adverse effect levels (NOAEL) and the inhibitory concentrations at 50% (IC50), which we reported previously for cinnamaldehyde. 18 The NOAEL values for the hESC and hPF were 7.6×10 −6 and 3×10 −6 M, respectively, while the IC 50 values were 4×10 −5 and 3.7×10 −5 M. Identification and quantification of organic chemicals using GC/MS After dilution with acetonitrile (Fisher Scientific, Fair Lawn, New Jersey, USA), refill fluids were analysed by GC/MS. Using internal standard-based calibration procedures similar to those described elsewhere, 19 analyses were performed with an Agilent (Santa Clara, California, USA) 7693 autosampler, Agilent 7890A GC, and Agilent 5975C MS. A DB-VRX phase GC capillary column was used (60 m×250 mm×1.4 mm film). For each replicate sample, 50 mL of each fluid was dissolved in 1 mL of acetonitrile, and 1 mL was then injected into the GC with a 10:1 split. The GC temperature programme for all analyses was as follows: 45°C hold for 5 min; 12°C/min to 189°C; hold at 189°C for 2 min; then 5°C/min to 245°C and hold for 10 min at 245°C. The MS was operated at electron ionization mode. The ion source temperature was 250°C. The scan range was from 34 to 400 amu; for quantitation of each analyte, use of scan mode facilitated verification so that no co-eluting peaks were affecting the results. Each target analyte was quantitated using authentic standard material, and an internal standard (1,2,3-trichlorobenzene) normalised non-linear multipoint calibration curve based on peak area. The quantitation ion for cinnamaldehyde was 131. The electron multiplier voltage was 1350 V. Cinnamon Ceylon aerosols with 3 and 5 V were collected in 0.4 mL water and added into 0.8 mL of acetonitrile before GC/MS analysis; with an injection of only 1 mL, and the injector split, the water caused no problems in the analyses. Concentrations of the chemicals detected in the 5 Vaerosol were estimated by accounting for dilutions into water and acetonitrile and using the amount of fluid consumed by weighing the tank before and after aerosol production. E-cigarette aerosols E-cigarette aerosols were produced with fresh unused cartomizers or tanks using a smoking machine. 2 20 21 The Vea cartomizer device and unfilled cartomizers (Johnson Creek, Hartland, Wisconsin) operated at 2.9 V, 2.1 Ω and 4 W. Cartomizers were loaded with 1 mL of refill fluid as recommended by the vendor and used in a manner that avoided dry puffing. An Innokin iTaste MVP 3.0 battery with variable voltage and wattage and Innokin iClear 16D bottom dual coil clearomizers (tanks) were operated at 3 V, 2.1 Ω and 4.2 W or at 5 V, 2.1 Ω and 11.9 W. For each sample, 2 mL of fluid was pipetted into new clean tanks. Puff duration was 4.3 s, the average for e-cigarette users, 22 and flow rate was adjusted to produce consistent robust puffs (eg, cartomizer 30 mL puffs and tank 56 mL puffs). Aerosols were collected in a round-bottom flask containing culture medium and submerged in an ice bath or dry ice bath. Aerosol solutions were made up to six total puff equivalents (TPE), where TPE are the number of puffs fully dissolved in 1 mL of culture medium. For the ice bath method, 12 puffs were collected in 2 mL of medium, while in the dry ice bath method, 24 puffs were collected into 4 mL of medium. Culturing hPF, A549 and hESC hPF were chosen as a differentiated adult lung cell that is often more sensitive to e-cigarette products than lung epithelium (unpublished data). hPF (ScienCell, Carlsbad, California, USA) were cultured on poly-L-lysine-coated flasks and dishes using the manufacturer’s protocol in complete fibroblast medium containing 2% fetal bovine serum, 1% fibroblast growth serum and 1% penicillin/streptomycin. 18 In experiments, hPF were dispersed into single cells and plated at a density of 4000 cells/0.32 cm 2 using a BioMate 3S Spectrophotometer (Thermo Fisher Scientific, Chino, California, USA)-based standard curve. A549 CCL-185 cells (ATCC, Manassas, Virginia, USA), a line of lung epithelial cells often used in toxicological testing, were cultured using the distributors’ protocol in ATCC F-12K Medium and 10% fetal bovine serum on tissue culture flasks. In experiments, cells at 80% confluency were rinsed in 0.25% trypsin and plated as single cells at a density of 50 000 cells/ 0.32 cm 2 using a BioMate 3S Spectrophotometer-based standard curve. hESC were used as a model for early postimplantation human embryos. hESC (H9) (WiCell, Madison, Wisconsin, USA) were cultured on Matrigel in mTeSR1 medium in six-well plates. 23 24 For experiments, wells at 60–80% confluency were washed with Dulbecco’s phosphate-buffered saline, and cells were enzymatically detached using Accutase (eBioscience, San Diego, California, USA). Large cell clumps were mechanically dispersed with sterile glass beads to form small colonies of 2–10 cells. For MTT experiments, cell concentration was adjusted using a BioMate 3S Spectrophotometer to produce 40 000 cells/ 0.32 cm 2 . 23 25 Cytotoxicity in the MTT assay Dose–response experiments using the MTT assay were performed using aerosols made from Cinnamon Ceylon refill fluid and a laboratory-made refill fluid containing cinnamaldehyde plus propylene glycol. Cinnamon Ceylon was used as a representative cinnamon flavour with high toxicity. Aerosols were tested at 0.06, 0.2, 0.6, 2 and 6 TPE. Cells were seeded in 96-well plates containing control wells, vapour effect control wells (wells adjacent to the highest concentration to ensure vapours did not affect neighbouring wells) and treatment wells. 25 MTT reagent was added after 48 hours of exposure, and 2 hours later MTT solution was added to the medium. Formazan crystals were solubilised in dimethyl sulfoxide, and absorbance was read at 570 nm. For each variable tested, three independent experiments were performed. Recovery experiments The hPF and hESC were plated in wells containing medium or medium with cinnamaldehyde (MTT IC 50 concentration). Periodically a well containing medium with cinnamaldehyde was washed with PBS, and fresh medium without cinnamaldehyde was added. This procedure was repeated hourly for 3 hours for hPF and every 2 hours for 8 hours for hESC. Cells were imaged after 24 and 48 hours to observe recovery. For each cell type, three independent experiments were performed. Effect on cytoskeleton The hPF and hESC were plated in chamber slides for 40 hours and then treated for 2 hours at control, MTT NOAEL and MTT IC 50 concentrations. After treatment, cells were fixed with 4% paraformaldehyde and blocked in goat serum for 30 min at room temperature. Cells were labelled using phalloidin-Alexa 488 and a β-tubulin TRITC-conjugated antibody. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Phase and fluorescent images were taken on a Nikon Eclipse inverted microscope. Four independent labelling experiments were performed.

Live cell imaging assay

When hESC cultures reach 80% confluency, colonies were passaged using ReLeSR (StemCell Technologies, Vancouver, Canada). In total, 600 colonies containing 10–20 cells were allowed to attach overnight. Colonies were treated with 7.6×10 −6 M of cinnamaldehyde. Time-lapse phase contrast images of control and treated cells were taken every hour for 70 hours in a Nikon BioStation CT using 3×3 tiling. Videos were generated and analysed using StemCellQC video bioinformatics software. 26 Totally, 10–15 colonies/group were analysed in three independent experiments.

Alkaline comet assay

Comet assays were performed to determine if cinnamaldehyde induced strand breaks in DNA. A549 cells and hPF were cultured 48 hours then treated for 3 hours using 3×10 −6 M cinnamaldehyde (MTT NOAEL concentration for hPF). 18 One group of hESC were treated with the MTT NOAEL concentration for 3 hours, while a second treated group was allowed to recover for 24 hours after treatment. Cells were harvested, suspended in agarose, lysed, subjected to alkaline electrophoresis (Trevigen) and stained with SYBR green. Fluorescent images were taken using an inverted microscope, and the percentage of cells with comet tails, comet tail length and olive moment (tail length×-fraction of DNA in tail) were determined with CometScore (Sumerduck, Virginia, USA). Single cells from 12 images were used to determine the percentage of cells with comet tails, and 100 cells/group were evaluated to determine comet tail length and olive moment. Three independent experiments were performed with each cell type.

Data analysis

For dose–response experiments, IC 50 values were computed with Prism software (GraphPad, San Diego, California, USA) using the log inhibitor vs normalised response-variable slope. An analysis of variance (ANOVA) on three independent experiments of the dose–response MTT and hESC comet assay data were performed using Graph Pad Prism. When significance was found, treated groups were compared to the lowest concentration using Dunnett’s post hoc test, and means were considered significantly different for p