Public Health Consequences of E-Cigarettes (2018) / Chapter Skim
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Appendix D: Cytotoxicity Tables
Appendix D: Cytotoxicity Tables
Pages 703-740
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D Cytotoxicity Tables This appendix contains summary tables (Tables D-1, D-2, and D-3) of in vitro studies in which cytotoxicity is assessed.
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, as an as an Cigarette Smoke Compares Cell or Tissue Type Used and Reference E-liquid (L) Exposure Exposure as a Control Flavors Comments Aufderheide A ü • mmortalized primary NHBE cell I and Emura, line (CL-1548)
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• uman lung epithelial carcinoma H cells (A549) continued 705
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, as an as an Cigarette Smoke Compares Cell or Tissue Type Used and Reference E-liquid (L) Exposure Exposure as a Control Flavors Comments Farsalinos et A ü ü ü ü • Monolayer-cultured cardiomyoblast al., 2013 cells (H9c2)
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However, fibroblasts are considered to be mesenchymal stem cells because of their self-renewing and multipotent character. Sancilio et al., L ü • HGF 2017 Scheffler et al., A ü ü • rimary NHBE cells P 2015a • uman lung epithelial carcinoma H cells (A549)
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, as an as an Cigarette Smoke Compares Cell or Tissue Type Used and Reference E-liquid (L) Exposure Exposure as a Control Flavors Comments Scheffler et al., A ü ü ü • rimary NHBE cells P 2015b • rimary cells came from two P donors (cells named NHBE48 and NHBE33)
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NOTE: hESC = human embryonic stem cell; HFL1 = human fetal lung fibroblast; HGF = human gingival fibroblast; HNSCC = head and neck squamous cell carcinoma; HPdLF = human periodontal ligament fibroblast; hPF = human pulmonary fibroblast; hTBE = human tracheobronchial epithelial; HUVEC = human umbilical vein endothelial cell; mNSC = mouse neural stem cell; NHBE = normal human bronchial epithelial.
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Bahl et al., 2012 • 5 different flavors 3 • hESC • 6 concentrations: • MTT assay • mNSC 0.001%, 0.01%, 0.03%, • hPF 0.1%, 0.3%, and 1% • ncubation at 37ºC, 5% I CO2, and 95% relative humidity for 48 hours Barber et al., 2016 • Combustible • HUVECs • 8-hour exposure to 4 • Endothelial cell tobacco cigarette the extracts viability, density and smoke extracts metabolic activity from Marlboro 100 after exposure to cigarettes (16 mg tar, mainstream and 1.2 mg/ml nicotine, sidestream tobacco Philip Morris) smoke extracts, e-aerosol extracts, and pure nicotine
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• E-cigarette aerosol extract from NJoy • Activation/deposition OneJoy device, of complement traditional flavor proteins onto with 1.2% (12 mg/ endothelial cells ml) or 1.8% (18 mg/ was quantified as a ml)
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Creek, Hartland, DAPI refill fluids • hESC Wisconsin) operated • ive cell imaging assay L • Aerosols produced at 2.9 V, 2.1 Ω, and 4 • lkaline comet assay A from the refill fluids W
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. cigarette and leading • Combustible tobacco the smoke stream over cigarette smoke the cell cultures for 50 (United Kingdom minutes.
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• he medium was T • 20 commercially aspirated and replaced available e-liquids by medium containing (17 tobacco flavored, the combustible 3 sweet or fruit tobacco smoke and flavored) , with 6–24 e-cigarette liquid mg/ml nicotine, extracts in one manufactured or undiluted (100%)
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. combustible tobacco Images were taken 24 cigarettes (University hours post-treatment.
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e-cigarette products. • Reference 3R4F combustible tobacco cigarettes (University of Kentucky)
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a 50/50 mixture of PG/glycerol. • hree battery output T voltage settings were tested: 3.3, 4.0, and 4.8 V
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flavor containing 16 • FL1 was treated H mg nicotine) with the following • SE from a research C e-liquids: PG, glycerol, grade combustible Vape Dudes (classic tobacco cigarette tobacco with or without nicotine)
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with and without nicotine and two market flavors (classic tobacco and magnificent menthol) • Reference 3R4F, 1R5F, and Marlboro gold combustible tobacco cigarettes continued 719
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Romagna et al., 2013 • Combustible tobacco • Mouse BALB/3T3 • E-cigarette aerosol and • MTT assay cigarette smoke fibroblasts combustible tobacco extract cigarette smoke extracts • 21 different simulating e-cigarette e-cigarette liquids. use added to culture Composition of medium.
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Cell medium fluorescence for mg/ml) from was replaced every 24 reactive oxygen species Halo Company hours.
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, warmed and type I concentration and natural and not warmed before in supernatants was artificial flavorings administration assayed using an (concentrations • GFs treated with H ELISA not provided by the fluid without • estern blot for LC3 W the manufacturer) , nicotine diluted 24 expression in HGF diluted 24 times with times, warmed and DMEM not warmed before administration • GFs also left H untreated Scheffler et al., 2015a • Reevo Mini-S • rimary NHBE cells P • he e-cigarette was T • ROS-Glo™ H2O2 Assay e-cigarette (In-Smoke, • Human lung connected to the piston (Promega, Madison, Winnenden, Germany)
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. • Reference 3R4F combustible tobacco cigarettes (Kentucky)
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• Cultures were • Combustible tobacco analyzed 24 hours cigarette smoke from after exposure. 10 reference K3R4F combustible tobacco cigarettes (Lexington, Kentucky)
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Welz et al., 2016 • -liquids in three E • Spheroidal cultures • 24-hour one-time • MTT assay flavors (apple, of oropharyngeal incubation cherry, and tobacco) mucosa • .5-hour incubation on 2 with a base mixture 5 sequential days of 80% PG, 10% glycerol, and 10% water and 12 mg/ml nicotine Willershausen et al., • E-liquids • Clonetics® HPdLF • p to 96-hour U • PrestoBlue Cell 2014 (eSmokerShop, incubation depending Viability Assay GmbH, Hannover, on assay • ATP detection Germany)
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• aCaT cells were H treated for 10 days, and UMSCC10B and HN30 for 12 days prior to colony counting. NOTE: 2-MOCA = 2-methoxycinnamaldehyde; CSE = cigarette smoke extract; DAPI = 4',6-diamidino-2-phenylindole; GC/MS = gas chromatography/mass spectrometry; hESC = human embryonic stem cell; HFL1 = human fetal lung fibroblast; HGF = human gingival fibroblast; HNSCC = head and neck squamous cell carcinoma; HPdLF = human periodontal ligament fibroblast; hPF = human pulmonary fibroblast; HRV = human rhinovirus; hTBE = human tracheobronchial epithelial; HUVEC = human umbilical vein endothelial cell; LDH = lactate dehydrogenase; mNSC = mouse neural stem cell; MTT = 3-(4,5-dimethylthiazol-2-yl)
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Authors observed a significant decrease in metabolic activity for cells that were exposed to combustible tobacco cigarette smoke or e-cigarette aerosol extracts, independent of the formulation of the extract. Exposure to pure nicotine did not alter endothelial cell metabolic activity.
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These changes in bioluminescence expression indicate potential cellular damage, such as DNA damage, oxidative stress, ion homeostasis, and membrane damage. Both e-liquid and aerosol exposure resulted in cellular damage, but e-cigarette aerosol exposure showed damage without significant growth inhibition.
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and progressive decrease in cell viability and increased LDH release. The authors observed a similar trend during the different time points in both cell lines, but keratinocytes appeared more susceptible to combustible tobacco cigarette smoke–induced toxicity after 24 hours.
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IC50 could be determined only for combustible tobacco cigarette smoke extract and for El Toro cigarrillos and El Toro puros, since for every other e-cigarette sample, viability was higher than 50% at all extract concentrations. The lowest NOAEL and IC50 were observed in combustible tobacco cigarette smoke extract.
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E-cigarette aerosols with coffee and strawberry flavors also significantly increased cytokine levels compared to both air controls and reference combustible tobacco cigarettes. No significant differences (p < 0.05)
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The IL-8 levels were all significantly increased in cells exposed to e-cigarette aerosol compared with the air controls. In cells exposed to e-cigarette aerosols, small but significant increases in fluorescence were observed.
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. Any IL-8 release from blu MM e-liquid treatments that were significant when compared with IL-8 release from exposure to combustible tobacco cigarettes occurred at doses approximately 42 fold higher than the combustible tobacco cigarettes.
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Combustible tobacco cigarette smoke extract exhibited significant cytotoxicity at extract concentrations greater than 12.5%. For the majority of e-liquids (13 of 21)
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. The reactive oxygen species production showed a peak after 24 hours of treatment compared with untreated controls (771.6 [nicotine]
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The viability of combustible tobacco cigarette mainstream smoke–exposed cells was 4.5–8 times lower and the oxidative stress levels 4.5–5 times higher than those of e-cigarette aerosol–exposed cells, depending on the donor. Welz et al., Both fruit- and tobacco-flavored extracts were cytotoxic to 2016 oropharyngeal tissue, but fruit-flavored liquids showed a higher toxicity than tobacco-flavored ones.
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led to a statistically significant reduction of ATP detection. The untreated human periodontal ligament fibroblasts and those incubated for 24 hours with PG showed good proliferation.
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NOTE: 2-MOCA = 2-methoxycinnamaldehyde; CSE = cigarette smoke extract; hESC = human embryonic stem cell; HFL1 = human fetal lung fibroblast; HGF = human gingival fibroblast; HNSCC = head and neck squamous cell carcinoma; HPdLF = human periodontal ligament fibroblast; hPF = human pulmonary fibroblast; HRV = human rhinovirus; hTBE = human tracheobronchial epithelial; HUVEC = human umbilical vein endothelial cell; LDH = lactate dehydrogenase; mNSC = mouse neural stem cell; MTT = 3-(4,5- imethylthiazol-2-yl d ) -2,5-diphenyltetrazolium bromide; NHBE = normal human bronchial epithelial; NOAEL = no observed adverse effect level; PG = propylene glycol.
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2015. Vapors produced by electronic cigarettes and e-juices with flavorings in duce toxicity, oxidative stress, and inflammatory response in lung epithelial cells and in mouse lung.
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2015b. Evaluation of e-cigarette liquid vapor and mainstream cigarette smoke after direct exposure of primary human bronchial epithelial cells.
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