Flavored e-cigarette liquids and cinnamaldehyde impair respiratory innate immune cell function

American Journal of Physiology - Lung Cellular and Molecular Physiology

Volumn 313, Issue 2, 2017, Pages L278-L292

  Clapp, Phillip W ^a   Pawlak, Erica A ^a   Lackey, Justin T ^b   Keating, James E ^b   Reeber, Steven L ^b   Glish, Gary L ^b   Jaspers, Ilona ^a  

^a UNIVERSITY OF NORTH CAROLINA SCHOOL OF MEDICINE   (United States)

^b University of North Carolina at Chapel Hill   (United States)

Author keywords

Cinnamaldehyde; e cigarette; e liquid; Innate immune cell function

Indexed keywords

CINNAMALDEHYDE; DITHIOTHREITOL; FLAVORING AGENT; INTERLEUKIN 6; INTERLEUKIN 8; NICOTINE; ACROLEIN;

ADULT; AEROSOL; ARTICLE; CELL FUNCTION; CELL NUCLEUS MEMBRANE; CELL VIABILITY; CHEMICAL COMPOSITION; CYTOKINE PRODUCTION; CYTOKINE RELEASE; CYTOPLASM; DOSE RESPONSE; ELECTRONIC CIGARETTE; EXTRACELLULAR TRAP; FEMALE; HUMAN; HUMAN CELL; INNATE IMMUNITY; LUNG ALVEOLUS MACROPHAGE; MASS SPECTROMETRY; NATURAL KILLER CELL; NEUTROPHIL; PHAGOCYTOSIS; PRIORITY JOURNAL; ADOLESCENT; ADVERSE EFFECTS; ANALOGS AND DERIVATIVES; CHEMICALLY INDUCED; DRUG EFFECTS; INFLAMMATION; MACROPHAGE; MALE; MIDDLE AGED; YOUNG ADULT;

ACROLEIN; ADOLESCENT; ADULT; ELECTRONIC CIGARETTES; FEMALE; HUMANS; IMMUNITY, INNATE; INFLAMMATION; KILLER CELLS, NATURAL; MACROPHAGES; MALE; MIDDLE AGED; NEUTROPHILS; NICOTINE; PHAGOCYTOSIS; YOUNG ADULT;

EID: 85026742913     PISSN: 10400605     EISSN: 15221504     Source Type: Journal    
DOI: 10.1152/ajplung.00452.2016     Document Type: Article

References (78)

1. Allen JG, Flanigan SS, LeBlanc M, Vallarino J, MacNaughton P, Stewart JH, Christiani DC. Flavoring chemicals in e-cigarettes: diacetyl, 2,3-pentanedione, and acetoin in a sample of 51 products, including fruit-, candy-, and cocktail-flavored e-cigarettes. Environ Health Perspect 124: 733–739, 2016. doi:10.1289/EHP348.
2. Amrock SM, Zakhar J, Zhou S, Weitzman M. Perception of e-cigarette harm and its correlation with use among U.S. adolescents. Nicotine Tob Res 17: 330–336, 2015. doi:10.1093/ntr/ntu156.
3. Aoshiba K, Nagai A, Yasui S, Konno K. Nicotine prolongs neutrophil survival by suppressing apoptosis. J Lab Clin Med 127: 186–194, 1996. doi:10.1016/S0022-2143(96)90077-3.
4. Arrazola RA, Singh T, Corey CG, Husten CG, Neff LJ, Apelberg BJ, Bunnell RE, Choiniere CJ, King BA, Cox S, McAfee T, Caraballo RS; Centers for Disease Control and Prevention (CDC). Tobacco use among middle and high school students—United States, 2011–2014. MMWR Morb Mortal Wkly Rep 64: 381–385, 2015.
5. Bahl V, Lin S, Xu N, Davis B, Wang YH, Talbot P. Comparison of electronic cigarette refill fluid cytotoxicity using embryonic and adult models. Reprod Toxicol 34: 529–537, 2012. doi:10.1016/j.reprotox.2012. 08.001.
6. Bansal V, Kim KH. Review on quantitation methods for hazardous pollutants released by e-cigarette (EC) smoking. Trends Anal Chem 78: 120–133, 2016. doi:10.1016/j.trac.2016.02.015.
7. Barrington-Trimis JL, Samet JM, McConnell R. Flavorings in electronic cigarettes: an unrecognized respiratory health hazard? JAMA 312: 2493–2494, 2014. doi:10.1001/jama.2014.14830.
8. Behar RZ, Davis B, Wang Y, Bahl V, Lin S, Talbot P. Identification of toxicants in cinnamon-flavored electronic cigarette refill fluids. Toxicol In Vitro 28: 198–208, 2014. doi:10.1016/j.tiv.2013.10.006.
9. Behar RZ, Luo W, Lin SC, Wang Y, Valle J, Pankow JF, Talbot P. Distribution, quantification and toxicity of cinnamaldehyde in electronic cigarette refill fluids and aerosols. Tob Control 25, Suppl 2: ii94–ii102, 2016. doi:10.1136/tobaccocontrol-2016-053224..
10. Brinkmann V, Laube B, Abed UA, Goosmann C, Zychlinsky A. Neutrophil extracellular traps: how to generate and visualize them. J Vis Exp pii: 1724, 2010. doi:10.3791/1724.
11. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A. Neutrophil extracellular traps kill bacteria. Science 303: 1532–1535, 2004. doi:10.1126/science.1092385.
12. Brinkmann V, Zychlinsky A. Beneficial suicide: why neutrophils die to make NETs. Nat Rev Microbiol 5: 577–582, 2007. doi:10.1038/nrmicro1710.
13. Brinkmann V, Zychlinsky A. Neutrophil extracellular traps: is immunity the second function of chromatin? J Cell Biol 198: 773–783, 2012. doi:10.1083/jcb.201203170.
14. Chao LK, Hua KF, Hsu HY, Cheng SS, Lin IF, Chen CJ, Chen ST, Chang ST. Cinnamaldehyde inhibits pro-inflammatory cytokines secretion from monocytes/macrophages through suppression of intracellular signaling. Food Chem Toxicol 46: 220–231, 2008. doi:10.1016/j.fct.2007.07.016.
15. Farsalinos KE, Romagna G, Tsiapras D, Kyrzopoulos S, Spyrou A, Voudris V. Impact of flavour variability on electronic cigarette use experience: an internet survey. Int J Environ Res Public Health 10: 7272–7282, 2013. doi:10.3390/ijerph10127272.
16. Farsalinos KE, Voudris V, Poulas K. E-cigarettes generate high levels of aldehydes only in ‘dry puff’ conditions. Addiction 110: 1352–1356, 2015. doi:10.1111/add.12942.
17. Ferson M, Edwards A, Lind A, Milton GW, Hersey P. Low natural killer-cell activity and immunoglobulin levels associated with smoking in human subjects. Int J Cancer 23: 603–609, 1979. doi:10.1002/ijc.2910230504.
18. Finkelstein EI, Ruben J, Koot CW, Hristova M, van der Vliet A. Regulation of constitutive neutrophil apoptosis by the α,β-unsaturated aldehydes acrolein and 4-hydroxynonenal. Am J Physiol Lung Cell Mol Physiol 289: L1019–L1028, 2005. doi:10.1152/ajplung.00227.2005.
19. Flavor and Extract Manufacturing Association of the United States. Respiratory Health and Safety in the Flavor Manufacturing Workplace. Washington, DC: FEMA, 2012. https://www.femaflavor.org/sites/default/files/linked_files/FEMA_2012%20Respiratory%20Health%20and%20 Safety.pdf [accessed 22 June 2017].
20. Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, Weinrauch Y, Brinkmann V, Zychlinsky A. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 176: 231–241, 2007. doi:10.1083/jcb.200606027.
21. Garvy BA, Harmsen AG. The importance of neutrophils in resistance to pneumococcal pneumonia in adult and neonatal mice. Inflammation 20: 499–512, 1996. doi:10.1007/BF01487042.
22. Geiss O, Bianchi I, Barrero-Moreno J. Correlation of volatile carbonyl yields emitted by e-cigarettes with the temperature of the heating coil and the perceived sensorial quality of the generated vapours. Int J Hyg Environ Health 219: 268–277, 2016. doi:10.1016/j.ijheh.2016.01.004.
23. Gerloff J, Sundar IK, Freter R, Sekera ER, Friedman AE, Robinson R, Pagano T, Rahman I. Inflammatory response and barrier dysfunction by different e-cigarette flavoring chemicals identified by gas chromatog-raphy-mass spectrometry in e-liquids and e-vapors on human lung epithelial cells and fibroblasts. Appl In Vitro Toxicol 3: 28–40, 2017. doi:10. 1089/aivt.2016.0030.
24. Ghio AJ, Bassett M, Chall AN, Levin DG, Bromberg PA. Bronchos-copy in healthy volunteers. J Bronchology Interv Pulmonol 5: 185–194, 1998.
25. Goniewicz ML, Knysak J, Gawron M, Kosmider L, Sobczak A, Kurek J, Prokopowicz A, Jablonska-Czapla M, Rosik-Dulewska C, Havel C, Jacob P 3rd, Benowitz N. Levels of selected carcinogens and toxicants in vapour from electronic cigarettes. Tob Control 23: 133–139, 2014. doi: 10.1136/tobaccocontrol-2012-050859.
26. Grana R, Benowitz N, Glantz SA. E-cigarettes: a scientific review. Circulation 129: 1972–1986, 2014. doi:10.1161/CIRCULATIONAHA. 114.007667.
27. Gray RD, Lucas CD, Mackellar A, Li F, Hiersemenzel K, Haslett C, Davidson DJ, Rossi AG. Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps. J Inflamm (Lond) 10: 12, 2013. doi:10.1186/1476-9255-10-12.
28. Gray RD, McCullagh BN, McCray PB Jr. NETs and CF lung disease: current status and future prospects. Antibiotics (Basel) 4: 62–75, 2015. doi:10.3390/antibiotics4010062.
29. Hahn J, Monakhova YB, Hengen J, Kohl-Himmelseher M, Schussler J, Hahn H, Kuballa T, Lachenmeier DW. Electronic cigarettes: overview of chemical composition and exposure estimation. Tob Induc Dis 12: 23, 2014. doi:10.1186/s12971-014-0023-6.
30. Harber P, Saechao K, Boomus C. Diacetyl-induced lung disease. Toxicol Rev 25: 261–272, 2006.
31. Holden VK, Hines SE. Update on flavoring-induced lung disease. Curr Opin Pulm Med 22: 158–164, 2016. doi:10.1097/MCP.0000000000000250.
32. Horvath KM, Brighton LE, Herbst M, Noah TL, Jaspers I. Live attenuated influenza virus (LAIV) induces different mucosal T cell function in nonsmokers and smokers. Clin Immunol 142: 232–236, 2012. doi:10.1016/j.clim.2011.12.013.
33. Horvath KM, Brighton LE, Zhang W, Carson JL, Jaspers I. Epithelial cells from smokers modify dendritic cell responses in the context of influenza infection. Am J Respir Cell Mol Biol 45: 237–245, 2011. doi:10.1165/rcmb.2010-0190OC.
34. Horvath KM, Herbst M, Zhou H, Zhang H, Noah TL, Jaspers I. Nasal lavage natural killer cell function is suppressed in smokers after live attenuated influenza virus. Respir Res 12: 102, 2011. doi:10.1186/1465-9921-12-102.
35. Hwang JH, Lyes M, Sladewski K, Enany S, McEachern E, Mathew DP, Das S, Moshensky A, Bapat S, Pride DT, Ongkeko WM, Crotty Alexander LE. Electronic cigarette inhalation alters innate immunity and airway cytokines while increasing the virulence of colonizing bacteria. J Mol Med (Berl) 94: 667–679, 2016. doi:10.1007/s00109-016-1378-3.
36. Jaspers I, Horvath KM, Zhang W, Brighton LE, Carson JL, Noah TL. Reduced expression of IRF7 in nasal epithelial cells from smokers after infection with influenza. Am J Respir Cell Mol Biol 43: 368–375, 2010. doi:10.1165/rcmb.2009-0254OC.
37. Jeyaseelan S, Young SK, Yamamoto M, Arndt PG, Akira S, Kolls JK, Worthen GS. Toll/IL-1R domain-containing adaptor protein (TIRAP) is a critical mediator of antibacterial defense in the lung against Klebsiella pneumoniae but not Pseudomonas aeruginosa. J Immunol 177: 538–547, 2006. doi:10.4049/jimmunol.177.1.538.
38. Jung HJ, Song YS, Kim K, Lim CJ, Park EH. Assessment of the anti-angiogenic, anti-inflammatory and antinociceptive properties of ethyl vanillin. Arch Pharm Res 33: 309–316, 2010. doi:10.1007/s12272-010-0217-2.
39. Khlystov A, Samburova V. Flavoring Compounds Dominate Toxic Aldehyde Production during E-Cigarette Vaping. Environ Sci Technol 50: 13080–13085, 2016. doi:10.1021/acs.est.6b05145.
40. Kim BH, Lee YG, Lee J, Lee JY, Cho JY. Regulatory effect of cinnamaldehyde on monocyte/macrophage-mediated inflammatory responses. Mediators Inflamm 2010: 529359, 2010. doi:10.1155/2010/529359.
41. Kim MC, Kim SJ, Kim DS, Jeon YD, Park SJ, Lee HS, Um JY, Hong S-H. Vanillic acid inhibits inflammatory mediators by suppressing NF-κB in lipopolysaccharide-stimulated mouse peritoneal macrophages. Immunopharmacol Immunotoxicol 33: 525–532, 2011. doi:10.3109/08923973.2010.547500.
42. Kosmider L, Sobczak A, Fik M, Knysak J, Zaciera M, Kurek J, Goniewicz ML. Carbonyl compounds in electronic cigarette vapors: effects of nicotine solvent and battery output voltage. Nicotine Tob Res 16: 1319–1326, 2014. doi:10.1093/ntr/ntu078.
43. Kreiss K, Gomaa A, Kullman G, Fedan K, Simoes EJ, Enright PL. Clinical bronchiolitis obliterans in workers at a microwave-popcorn plant. N Engl J Med 347: 330–338, 2002. doi:10.1056/NEJMoa020300.
44. Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, McIntosh S, Robinson R, Rahman I. Vapors produced by electronic cigarettes and e-juices with flavorings induce toxicity, oxidative stress, and inflammatory response in lung epithelial cells and in mouse lung. PLoS One 10: e0116732, 2015. doi:10.1371/journal.pone.0116732.
45. Liao BC, Hsieh CW, Liu YC, Tzeng TT, Sun YW, Wung BS. Cinnamaldehyde inhibits the tumor necrosis factor-alpha-induced expression of cell adhesion molecules in endothelial cells by suppressing NF-kappaB activation: effects upon IkappaB and Nrf2. Toxicol Appl Pharmacol 229: 161–171, 2008. doi:10.1016/j.taap.2008.01.021.
46. Martin EM, Clapp PW, Rebuli ME, Pawlak EA, Glista-Baker E, Benowitz NL, Fry RC, Jaspers I. E-cigarette use results in suppression of immune and inflammatory-response genes in nasal epithelial cells similar to cigarette smoke. Am J Physiol Lung Cell Mol Physiol 311: L135–L144, 2016. doi:10.1152/ajplung.00170.2016.
47. Matthews JB, Chen FM, Milward MR, Wright HJ, Carter K, McDonagh A, Chapple IL. Effect of nicotine, cotinine and cigarette smoke extract on the neutrophil respiratory burst. J Clin Periodontol 38: 208–218, 2011. doi:10.1111/j.1600-051X.2010.01676.x.
48. McNeill A, Brose L, Calder R, Hitchman S, Hajek P, McRobbie H. E-cigarettes: An Evidence Update. A Report Commissioned by Public Health England. London, UK: Public Health England, 2015.
49. Mendes SJ, Sousa FI, Pereira DM, Ferro TA, Pereira IC, Silva BL, Pinheiro AJ, Mouchrek AQ, Monteiro-Neto V, Costa SK, Nascimento JL, Grisotto MA, da Costa R, Fernandes ES. Cinnamaldehyde modulates LPS-induced systemic inflammatory response syndrome through TRPA1-dependent and independent mechanisms. Int Immunopharmacol 34: 60–70, 2016. doi:10.1016/j.intimp.2016.02.012.
50. Müller L, Brighton LE, Jaspers I. Ozone exposed epithelial cells modify cocultured natural killer cells. Am J Physiol Lung Cell Mol Physiol 304: L332–L341, 2013. doi:10.1152/ajplung.00256.2012.
51. Murakami Y, Hirata A, Ito S, Shoji M, Tanaka S, Yasui T, Machino M, Fujisawa S. Re-evaluation of cyclooxygenase-2-inhibiting activity of vanillin and guaiacol in macrophages stimulated with lipopolysaccharide. Anticancer Res 27: 801–807, 2007.
52. Nauseef WM. How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev 219: 88–102, 2007. doi:10.1111/j.1600-065X.2007.00550.x.
53. Nguyen H, Finkelstein E, Reznick A, Cross C, van der Vliet A. Cigarette smoke impairs neutrophil respiratory burst activation by alde-hyde-induced thiol modifications. Toxicology 160: 207–217, 2001. doi:10. 1016/S0300-483X(00)00450-9.
54. Noah TL, Zhou H, Jaspers I. Alteration of the nasal responses to influenza virus by tobacco smoke. Curr Opin Allergy Clin Immunol 12: 24–31, 2012. doi:10.1097/ACI.0b013e32834ecc80.
55. Noah TL, Zhou H, Monaco J, Horvath K, Herbst M, Jaspers I. Tobacco smoke exposure and altered nasal responses to live attenuated influenza virus. Environ Health Perspect 119: 78–83, 2011. doi:10.1289/ehp.1002258.
56. Olmedo P, Navas-Acien A, Hess C, Jarmul S, Rule A. A direct method for e-cigarette aerosol sample collection. Environ Res 149: 151–156, 2016. doi:10.1016/j.envres.2016.05.008.
57. Pilsczek FH, Salina D, Poon KK, Fahey C, Yipp BG, Sibley CD, Robbins SM, Green FH, Surette MG, Sugai M, Bowden MG, Hussain M, Zhang K, Kubes P. A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol 185: 7413–7425, 2010. doi:10.4049/jimmunol.1000675.
58. Pokhrel P, Fagan P, Kehl L, Herzog TA. Receptivity to e-cigarette marketing, harm perceptions, and e-cigarette use. Am J Health Behav 39: 121–131, 2015. doi:10.5993/AJHB.39.1.13.
59. Rager JE, Bauer RN, Müller LL, Smeester L, Carson JL, Brighton LE, Fry RC, Jaspers I. DNA methylation in nasal epithelial cells from smokers: identification of ULBP3-related effects. Am J Physiol Lung Cell Mol Physiol 305: L432–L438, 2013. doi:10.1152/ajplung.00116.2013.
60. Romano B, Borrelli F, Fasolino I, Capasso R, Piscitelli F, Cascio M, Pertwee R, Coppola D, Vassallo L, Orlando P, Di Marzo V, Izzo A. The cannabinoid TRPA1 agonist cannabichromene inhibits nitric oxide production in macrophages and ameliorates murine colitis. Br J Pharmacol 169: 213–229, 2013. doi:10.1111/bph.12120.
61. Ryder MI. Nicotine effects on neutrophil F-actin formation and calcium release: implications for tobacco use and pulmonary diseases. Exp Lung Res 20: 283–296, 1994. doi:10.3109/01902149409064388.
62. Seow WK, Thong YH, Nelson RD, MacFarlane GD, Herzberg MC. Nicotine-induced release of elastase and eicosanoids by human neutrophils. Inflammation 18: 119–127, 1994. doi:10.1007/BF01534553.
63. Sherwood CL, Boitano S. Airway epithelial cell exposure to distinct e-cigarette liquid flavorings reveals toxicity thresholds and activation of CFTR by the chocolate flavoring 2,5-dimethypyrazine. Respir Res 17: 57, 2016. doi:10.1186/s12931-016-0369-9.
64. Stämpfli MR, Anderson GP. How cigarette smoke skews immune responses to promote infection, lung disease and cancer. Nat Rev Immunol 9: 377–384, 2009. doi:10.1038/nri2530.
65. Stockley JA, Walton GM, Lord JM, Sapey E. Aberrant neutrophil functions in stable chronic obstructive pulmonary disease: the neutrophil as an immunotherapeutic target. Int Immunopharmacol 17: 1211–1217, 2013. doi:10.1016/j.intimp.2013.05.035.
66. Sundar IK, Javed F, Romanos GE, Rahman I. E-cigarettes and flavorings induce inflammatory and pro-senescence responses in oral epithelial cells and periodontal fibroblasts. Oncotarget 7: 77196–77204, 2016. doi:10.18632/oncotarget.12857.
67. Sussan TE, Gajghate S, Thimmulappa RK, Ma J, Kim JH, Sudini K, Consolini N, Cormier SA, Lomnicki S, Hasan F, Pekosz A, Biswal S. Exposure to electronic cigarettes impairs pulmonary anti-bacterial and anti-viral defenses in a mouse model. PLoS One 10: e0116861, 2015. doi:10.1371/journal.pone.0116861.
68. Tateda K, Moore TA, Deng JC, Newstead MW, Zeng X, Matsukawa A, Swanson MS, Yamaguchi K, Standiford TJ. Early recruitment of neutrophils determines subsequent T1/T2 host responses in a murine model of Legionella pneumophila pneumonia. J Immunol 166: 3355– 3361, 2001. doi:10.4049/jimmunol.166.5.3355.
69. Tayyarah R, Long GA. Comparison of select analytes in aerosol from e-cigarettes with smoke from conventional cigarettes and with ambient air. Regul Toxicol Pharmacol 70: 704–710, 2014. doi:10.1016/j.yrtph.2014. 10.010.
70. Tollerud DJ, Clark JW, Brown LM, Neuland CY, Mann DL, Pankiw-Trost LK, Blattner WA, Hoover RN. Association of cigarette smoking with decreased numbers of circulating natural killer cells. Am Rev Respir Dis 139: 194–198, 1989. doi:10.1164/ajrccm/139.1.194.
71. Topham NJ, Hewitt EW. Natural killer cell cytotoxicity: how do they pull the trigger? Immunology 128: 7–15, 2009. doi:10.1111/j.1365-2567. 2009.03123.x.
72. Varlet V, Farsalinos K, Augsburger M, Thomas A, Etter JF. Toxicity assessment of refill liquids for electronic cigarettes. Int J Environ Res Public Health 12: 4796–4815, 2015. doi:10.3390/ijerph120504796.
73. Williams M, Villarreal A, Bozhilov K, Lin S, Talbot P. Metal and silicate particles including nanoparticles are present in electronic cigarette cartomizer fluid and aerosol. PLoS One 8: e57987, 2013. doi:10.1371/journal.pone.0057987.
74. World Health Organization. WHO Global Report on Mortality Attributable to Tobacco. Geneva, Switzerland: WHO, 2012.
75. Xu Y, Li H, Bajrami B, Kwak H, Cao S, Liu P, Zhou J, Zhou Y, Zhu H, Ye K, Luo HR. Cigarette smoke (CS) and nicotine delay neutrophil spontaneous death via suppressing production of diphosphoinositol pen-takisphosphate. Proc Natl Acad Sci USA 110: 7726–7731, 2013. doi:10. 1073/pnas.1302906110.
76. Yost CC, Cody MJ, Harris ES, Thornton NL, McInturff AM, Martinez ML, Chandler NB, Rodesch CK, Albertine KH, Petti CA, Weyrich AS, Zimmerman GA. Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates. Blood 113: 6419–6427, 2009. doi:10.1182/blood-2008-07-171629.
77. Youn HS, Lee JK, Choi YJ, Saitoh SI, Miyake K, Hwang DH, Lee JY. Cinnamaldehyde suppresses toll-like receptor 4 activation mediated through the inhibition of receptor oligomerization. Biochem Pharmacol 75: 494–502, 2008. doi:10.1016/j.bcp.2007.08.033.
78. Zhu SH, Sun JY, Bonnevie E, Cummins SE, Gamst A, Yin L, Lee M. Four hundred and sixty brands of e-cigarettes and counting: implications for product regulation. Tob Control 23, Suppl 3: iii3–iii9, 2014. doi:10. 1136/tobaccocontrol-2014-051670.