Driven by new regulatory demands to demonstrate risk reduction, the toxicity assessment of tobacco products increasingly employs innovative in vitro methods, including biphasic cell and tissue cultures exposed to whole cigarette smoke at the air–liquid interface, cell transformation assays, and genomic analyses. At the same time, novel tobacco products are increasingly compared to traditional cigarettes. This overview of in vitro toxicology studies of tobacco products reported in the last five years provides evidence to support the prioritisation of in vitro over in vivo methods by industry and their recommendation by regulatory authorities.
IOM (2012). Scientific Standards for Studies on Modified Risk Tobacco Products, 370pp. Washington, DC, USA: The National Academies Press.
2.
JohnsonM.D., SchilzJ., DjordjevicM.V., RiceJ.R. & ShieldsP.G. (2009). Evaluation of in vitro assays for assessing the toxicity of cigarette smoke and smokeless tobacco. Cancer Epidemiology, Biomarkers & Prevention18, 3263–3304.
3.
ThorneD. & AdamsonJ. (2013). A review of in vitro cigarette smoke exposure systems. Experimental & Toxicologic Pathology65, 1183–1193.
4.
AdamsonJ., ThorneD., ErringtonG., FieldsW., LiX., PayneR., KrebsT., DalrympleA., FowlerK., DillonD., XieF. & MeredithC. (2014). An inter-machine comparison of tobacco smoke particle deposition in vitro from six independent smoke exposure systems. Toxicology in Vitro28, 1320–1328.
5.
AdamsonJ., ThorneD., McAugheyJ., DillonD. & MeredithC. (2013). Quantification of cigarette smoke particle deposition in vitro using a triplicate quartz crystal microbalance exposure chamber. BioMed Research International2013, Article ID 685074.
6.
AufderheideM., SchefflerS., MöhleN., HalterB. & HochrainerD. (2011). Analytical in vitro approach for studying cyto- and genotoxic effects of particulate airborne material. Analytical & Bioanalytical Chemistry401, 3213–3220.
7.
RachJ., BuddeJ., MöhleN. & AufderheideM. (2013). Direct exposure at the air–liquid interface: Evaluation of an in vitro approach for simulating inhalation of airborne substances. Journal of Applied Toxicology34, 506–515.
8.
OliveraD., KnallC., BoggsS. & SeagraveJ. (2010). Cytoskeletal modulation and tyrosine phosphorylation of tight junction proteins are associated with mainstream cigarette smoke-induced permeability of airway epithelium. Experimental & Toxicologic Pathology62, 133–143.
9.
NaraH., FukanoY., NishinoT. & AufderheideM. (2013). Detection of the cytotoxicity of water-insoluble fraction of cigarette smoke by direct exposure to cultured cells at an air–liquid interface. Experimental & Toxicologic Pathology65, 683–688.
10.
ThorneD., KilfordJ., PayneR., HaswellL., DalrympleA., MeredithC. & DillonD. (2014). Development of a BALB/c 3T3 Neutral Red Uptake cytotoxicity test using a mainstream cigarette smoke exposure system. BMC Research Notes7, 367.
11.
LiX., ShangP., PengB., NieC., ZhaoL., LiuH. & XieJ. (2012). Effects of smoking regimens and test material format on the cytotoxicity of mainstream cigarette smoke. Food & Chemical Toxicology50, 545–551.
12.
ArimilliS., DamratoskiB.E., BombickB., BorgerdingM.F. & PrasadG.L. (2012). Evaluation of cytotoxicity of different tobacco product preparations. Regulatory Toxicology & Pharmacology64, 350–360.
13.
ChenH., CuiL., JiangX.Y., PangY.Q., TangG.L., HouH.W., JiangJ.H. & HuQ.Y. (2012). Evaluation of the cytotoxicity of cigarette smoke condensate by a cellular impedance biosensor. Food & Chemical Toxicology50, 612–618.
14.
RichterP.A., LiA.P., PolzinG. & RoyS.K. (2010). Cytotoxicity of eight cigarette smoke condensates in three test systems: Comparisons between assays and condensates. Regulatory Toxicology & Pharmacology58, 428–436.
15.
LouJ., ZhouG., ChuG., JiangJ., HuangF., ZhengS., LuY., LiX. & HeJ. (2010). Studying the cyto-genotoxic effects of 12 cigarette smoke condensates on human lymphoblastoid cell line in vitro. Mutation Research696, 48–54.
16.
MessnerB., FrotschnigS., Steinacher-NigischA., WinterB., EichmairE., GebetsbergerJ., SchwaigerS., PlonerC., LauferG. & BernhardD. (2012). Apoptosis and necrosis: Two different outcomes of cigarette smoke condensate-induced endothelial cell death. Cell Death & Disease3, e424.
17.
GaoH., PrasadG.L. & ZachariasW. (2013). Differential cell-specific cytotoxic responses of oral cavity cells to tobacco preparations. Toxicology in Vitro27, 282–291.
18.
CombesR., ScottK., DillonD., MeredithC., McAdamK. & ProctorC. (2012). The effect of a novel tobacco process on the in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter. Toxicology in Vitro26, 1022–1029.
19.
McAdamK.G., GreggE.O., LiuC., DittrichD.J., DukeM.G. & ProctorC.J. (2011). The use of a novel tobacco-substitute sheet and smoke dilution to reduce toxicant yields in cigarette smoke. Food & Chemical Toxicology49, 1684–1696.
20.
GaworskiC.L., SchramkeH., DiekmannJ., MeisgenT.J., TewesF.J., VeltelD.J., VanscheeuwijckP.M., RajendranN., MuzzioM. & HaussmannH.J. (2009). Effect of filtration by activated charcoal on the toxicological activity of cigarette mainstream smoke from experimental cigarettes. Inhalation Toxicology21, 688–704.
21.
ZenzenV., DiekmannJ., GerstenbergB., WeberS., WittkeS. & SchorpM.K. (2012). Reduced exposure evaluation of an electrically heated cigarette smoking system. Part 2: Smoke chemistry and in vitro toxicological evaluation using smoking regimens reflecting human puffing behavior. Regulatory Toxicology & Pharmacology64, S11–S34.
22.
FarsalinosK.E., RomagnaG., AllifranchiniE., RipamontiE., BocchiettoE., TodeschiS., TsiaprasD., KyrzopoulosS. & VoudrisV. (2013). Comparison of the cytotoxic potential of cigarette smoke and electronic cigarette vapour extract on cultured myocardial cells. International Journal of Environmental Research & Public Health10, 5146–5162.
23.
CervellatiF., MuresanX.M., SticozziC., GambariR., MontagnerG., FormanH.J., TorricelliC., MaioliE. & ValacchiG. (2014). Comparative effects between electronic and cigarette smoke in human keratinocytes and epithelial lung cells. Toxicology in Vitro28, 999–1005.
24.
KosmiderB., MessierE.M., ChuH.W. & MasonR.J. (2011). Human alveolar epithelial cell injury induced by cigarette smoke. PLoS One6, e26059.
25.
LeeI.T., WangS.W., LeeC.W., ChangC.C., LinC.C., LuoS.F. & YangC.M. (2008). Lipoteichoic acid induces HO-1 expression via the TLR2/MyD88/c-Src/NADPH oxidase pathway and Nrf2 in human tracheal smooth muscle cells. Journal of Immunology181, 5098–5110.
26.
YangG.Y., ZhangC.L., LiuX.C., QianG. & DengD.Q. (2013). Effects of cigarette smoke extracts on the growth and senescence of skin fibroblasts in vitro. International Journal of Biological Sciences9, 613–623.
27.
ScottK., SaulJ., CrooksI., CamachoO.M., DillonD. & MeredithC. (2013). The resolving power of in vitro genotoxicity assays for cigarette smoke particulate matter. Toxicology in Vitro27, 1312–1319.
28.
BrehenyD., CunninghamF., KilfordJ., PayneR., DillonD. & MeredithC. (2014). Application of a modified gaseous exposure system to the in vitro toxicological assessment of tobacco smoke toxicants. Environmental & Molecular Mutagenesis55, 662–672.
29.
Garcia-CantonC., ErringtonG., AnadonA. & MeredithC. (2014). Characterisation of an aerosol exposure system to evaluate the genotoxicity of whole mainstream cigarette smoke using the in vitro γH2AX assay by high content screening. BMC Pharmacology & Toxicology23, 15–41.
30.
GuoX., VerklerT.L., ChenY., RichterP.A., PolzinG.M., MooreM.M. & MeiN. (2011). Mutagenicity of 11 cigarette smoke condensates in two versions of the mouse lymphoma assay. Mutagenesis26, 273–281.
31.
OkuwaK., TanakaM., FukanoY., NaraH., NishijimaY. & NishinoT. (2010). In vitro micronucleus assay for cigarette smoke using a whole smoke exposure system: A comparison of smoking regimens. Experimental & Toxicologic Pathology62, 433–440.
32.
WeberS., HebestreitM., WilmsT., ConroyL.L. & RodrigoG. (2013). Comet assay and air–liquid interface exposure system: A new combination to evaluate genotoxic effects of cigarette whole smoke in human lung cell lines. Toxicology in Vitro27, 1987–1991.
33.
CavalloD., UrsiniC.L., FresegnaA.M., MaielloR., CiervoA., FerranteR., BurestiG. & IavicoliS. (2013). Cyto-genotoxic effects of smoke from commercial filter and non-filter cigarettes on human bronchial and pulmonary cells. Mutation Research750, 1–11.
34.
YaukC.L., WilliamsA., BuickJ.K., ChenG., MaertensR.M., HalappanavarS. & WhiteP.A. (2012). Genetic toxicology and toxicogenomic analysis of three cigarette smoke condensates in vitro reveals few differences among full-flavor, blonde, and light products. Environmental & Molecular Mutagenesis53, 281–296.
35.
CogginsC.R., BallantyneM., CurvallM. & RutqvistL.E. (2012). The in vitro toxicology of Swedish snus. Critical Reviews in Toxicology42, 304–313.
36.
MulliganR.M., AtkinsonC., VertegelA.A., ReukovV. & SchlosserR.J. (2009). Cigarette smoke extract stimulates interleukin-8 production in human airway epithelium and is attenuated by superoxide dismutase in vitro. American Journal of Rhinology & Allergy23, e1–e4.
37.
St-LaurentJ., ProulxL.I., BouletL.P. & BissonnetteE. (2009). Comparison of two in vitro models of cigarette smoke exposure. Inhalation Toxicology21, 1148–1153.
38.
WeisenseeD., PothA., RoemerE., ConroyL.L. & SchlageW.K. (2013). Cigarette smoke-induced morphological transformation of Bhas 42 cells in vitro. ATLA41, 181–189.
39.
TalikkaM., SierroN., IvanovN.V., ChaudharyN., PeckM.J., HoengJ., CogginsC.R. & PeitschM.C. (2012). Genomic impact of cigarette smoke, with application to three smoking-related diseases. Critical Reviews in Toxicology42, 877–889.
Gonzalez-SuarezI., SewerA., WalkerP., MathisC., EllisS., WoodhouseH., GuedjE., DulizeR., MarescottiD., AcaliS., MartinF., IvanovN.V., HoengJ. & PeitschM.C. (2014). Systems biology approach for evaluating the biological impact of environmental toxicants in vitro. Chemical Research in Toxicology27, 367–376.
42.
MathisC., PoussinC., WeisenseeD., GebelS., HengstermannA., SewerA., BelcastroV., XiangY., AnsariS., WagnerS., HoengJ. & PeitschM.C. (2013). Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air–liquid interface resemble bronchial epithelium from human smokers. American Journal of Physiology: Lung Cellular & Molecular Physiology304, L489–L503.
43.
SacksP.G., ZhaoZ.L., KosinskaW., FleisherK.E., GordonT. & GuttenplanJ.B. (2011). Concentration dependent effects of tobacco particulates from different types of cigarettes on expression of drug metabolizing proteins, and benzo(a)pyrene metabolism in primary normal human oral epithelial cells. Food & Chemical Toxicology49, 2348–2355.
44.
EskesC., SullivanK., AardemaM., SpielmannH., HillE., SchoetersG., CurrenR., VinkenM. & BlaauboerB. (2012). Comparing the challenges in developing and implementing 3Rs alternative methods in Europe and United States: Industrial and academic perspectives. ALTEX 1/12, WC8 Proceedings, 205–209.
45.
PiersmaA.H., EzendamJ., LuijtenM., MullerJ.J., RorijeE., van der VenL.T. & van BenthemJ. (2014). A critical appraisal of the process of regulatory implementation of novel in vivo and in vitro methods for chemical hazard and risk assessment. Critical Reviews in Toxicology44, 876–894.
46.
JenningsP. (2014). The future of in vitro toxicology. Toxicology in Vitro pii: S0887-2333(14) 00189-1. [E-pub ahead of print.]
47.
WilcoxN. & GoldbergA. (2011). Food for Thought… on validation. A puzzle or a mystery: An approach founded on new science. ALTEX28, 3–8.
48.
AnkleyG.T., BennettR.S., EricksonR.J., HoffD.J., HornungM.W., JohnsonR.D., MountD.R., NicholsJ.W., RussomC.L., SchmiederP.K., SerrranoJ.A., TietgeJ.E. & VilleneuveD.L. (2010). Adverse outcome pathways: A conceptual framework to support ecotoxicology research and risk assessment. Environmental Toxicology & Chemistry29, 730–741.
49.
LandesmannB., MennecozziM., BerggrenE. & WhelanM. (2013). Adverse outcome pathway-based screening strategies for an animal-free safety assessment of chemicals. ATLA41, 461–471.
OberdörsterG. & PottF. (1987). Extrapolation from rat studies with environmental tobacco smoke (ETS) to humans: Comparison of particle mass deposition and of clearance behavior of ETS compounds. Toxicology Letters35, 107–112.
52.
BrownJ.S., WilsonW.E. & GrantL.D. (2005). Dosimetric comparisons of particle deposition and retention in rats and humans. Inhalation Toxicology17, 355–385.
53.
CastellJ.V., DonatoM.T. & Gómez-LechónM.J. (2005). Metabolism and bioactivation of toxicants in the lung. The in vitro cellular approach. Experimental & Toxicologic Pathology57, Suppl. 1, 189–204.
54.
AbdoN., XiaM., BrownC.C., KosykO., HuangR., SakamuruS., ZhouY.H., JackJ., GallinsP., XiaK., LiY., ChiuW.A., Motsinger-ReifA., AustinC.P., TiceR.R., RusynI. & WrightF.A. (2015). Population-based in vitro hazard and concentration–response assessment of chemicals: The 1000 Genomes High-Throughput Screening Study. Environmental Health Perspectives [E-pub ahead of print.]
55.
PanL., YangX., LiS. & JiaC. (2015). Association of CYP2A6 gene polymorphisms with cigarette consumption: A meta-analysis. Drug & Alcohol Dependence [E-pub ahead of print.]
56.
PoveyA.C., MargisonG.P. & Santibáñez-KorefM.F. (2007). Lung cancer risk and variation in MGMT activity and sequence. DNA Repair6, 1134–1144.
57.
CrooksI., ScottK., DalrympleA., DillonD. & MeredithC. (2015). The combination of two novel tobacco blends and filter technologies to reduce the in vitro genotoxicity and cytotoxicity of prototype cigarettes. Regulatory Toxicology & Pharmacology [E-pub ahead of print.]
58.
LowY., SedykhA., FourchesD., GolbraikhA., WhelanM., RusynI. & TropshaA. (2013). Integrative chemical-biological read-across approach for chemical hazard classification. Chemical Research in Toxicology26, 1199–1208.
59.
MisraM., LeveretteR.D., CooperB.T., BennettM.B. & BrownS.E. (2014). Comparative in vitro toxicity profile of electronic and tobacco cigarettes, smokeless tobacco and nicotine replacement therapy products: E-liquids, extracts and collected aerosols. International Journal of Environmental Research & Public Health11, 11, 325–11, 347.
60.
FarsalinosK.E. & PolosaR. (2014). Safety evaluation and risk assessment of electronic cigarettes as tobacco cigarette substitutes: A systematic review. Therapeutic Advances in Drug Safety5, 67–86.
61.
OrrM.S. (2014). Electronic cigarettes in the USA: A summary of available toxicology data and suggestions for the future. Tobacco Control23, Suppl. 2, ii18–ii22.
62.
WilloughbyJ.A. (2014). Predicting respiratory toxicity using a human 3D airway (EpiAirway) model combined with multiple parametric analysis. Applied in Vitro Toxicology1, 21–31.
63.
SchlageW.K., IskandarA.R., KostadinovaR., XiangY., SewerA., MajeedS., KuehnD., FrentzelS., TalikkaM., GeertzM., MathisC., IvanovN., HoengJ. & PeitschM.C. (2014). In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures. Toxicology Mechanisms & Methods24, 470–487.
64.
JianlinL., GuohaiC., GuojunZ., JianJ., FangfangH., JuanjuanX., ShuZ., ZhijianC., WeiJ., YezhenL., XiaoxueL. & JiliangH. (2009). Studying the impact of S9 on cyto-genotoxicity of cigarette smoke in human peripheral blood lymphocytes in vitro. Environmental Toxicology & Pharmacology28, 275–279.
65.
LouJ., ChuG., ZhouG., JiangJ., HuangF., XuJ., ZhengS., JiangW., LuY., LiX., ChenZ. & HeJ. (2010). Comparison between two kinds of cigarette smoke condensates (CSCs) of the cytogenotoxicity and protein expression in a human B-cell lymphoblastoid cell line using CCK-8 assay, Comet assay and protein microarray. Mutation Research697, 55–59.
66.
ZhangJ., JuedesN., NarayanV.M., YueB., RockwoodA.L., PalmaN.L. & PatelJ.M. (2010). A cellular model to mimic exhaled cigarette smoke induced lung microvascular endothelial cell injury and death. International Journal of Clinical & Experimental Medicine3, 223–232.
67.
CombesR., ScottK., CrooksI., DillonD., MeredithC., McAdamK. & ProctorC. (2013). The in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter with reduced toxicant yields. Toxicology in Vitro27, 1533–1541.
68.
JianlinL., GuohaiC., GuojunZ., JianJ., FangfangH., JuanjuanX., ShuZ., ZhijianC., WeiJ., YezhenL., XiaoxueL. & JiliangH. (2009). Assessing cytogenotoxicity of cigarette smoke condensates using three in vitro assays. Mutation Research677, 21–26.
