Caner GEYİK, Z. Pinar GUMUS, Gorkem YARARBAS

COMPOSITION ANALYSIS OF E-LIQUIDS AND THEIR EFFECTS ON HEALTHY LIVER AND PHARYNGEAL CARCINOMA CELL LINES

Henüz sağlık üzerindeki etkileri tam olarak bilinmese de elektronik sigaralar, son yıllarda dünya çapında popüler hale gelmiştir. Yasal düzenlemelerin eksikliği, beraberinde ürün etiketinde yazan ve gerçekten içerikte bulunan maddeler arasında bir tutarsızlık olmasına sebep olmaktadır. Bu çalışmada, yaygın olarak kullanılan e-sıvıların içeriklerini ve iki farklı hücre hattı üzerindeki etkilerini analiz etmeyi amaçladık. Erişilebilirlik ve popülerliklerine göre on bir e-sıvı numunesi seçildi. E-sıvılardaki nikotin, propilen glikol (PG), gliserin (GLY) ve uçucu bileşikler, yüksek performanslı sıvı kromatografisi (HPLC) ve gaz kromatografisi (GC) kullanılarak analiz edildi. E-sıvıların, sağlıklı karaciğer hücre hattı THLE-2 ve faringeal karsinom hücre hattı Detroit 562 modelleri üzerinde hücre canlılığına etkisi, 3-(4,5-dimetiltiyazolil-2)-2,5-difeniltetrazolyum bromür (MTT) canlılık testi kullanılarak belirlendi. Nikotin miktarlarının ürün etiketleriyle uyumlu olduğu bulundu. PG ve GLY ise sadece markalar arasında değil aynı marka içindeki ürünler için de farklılık gösterdi. THLE-2 hücre canlılığı, e-sıvı konsantrasyonu ile ters orantılı olarak bulundu. Bununla beraber, hücre canlılığındaki azalmalar nikotin miktarı ile ilişkili değildi. İlginç bir şekilde, bazı numunelerin Detroit 562 hücreleri üzerindeki etkileri, düşük dozlarda canlılıkta azalma, orta konsantrasyonlarda hücre hayatta kalması ve en yüksek dozlarda canlılık kaybı olmak üzere üç fazlı olarak gözlemlendi. E-sıvıların analitik bileşimi, farklı hücresel etkilere karşılık gelen ürünler arasında büyük farklılıklar göstermektedir. Kanser hücrelerinin canlılığının doza bağlı bir şekilde değişmemesi, bu ürünlerin sonuçlarında hücresel farklılıkların rol oynadığını düşündürmektedir.

COMPOSITION ANALYSIS OF E-LIQUIDS AND THEIR EFFECTS ON HEALTHY LIVER AND PHARYNGEAL CARCINOMA CELL LINES

Electronic cigarettes have become popular worldwide in recent years although their effects on human health are still not properly known. The lack of regulations brings a problem of inconsistency between ingredients and the product label. We aimed to analyse the contents of widely used e-liquids and their effects on two different cell lines. Eleven e-liquid samples were selected according to their availability and popularity. Nicotine, propylene glycol (PG), glycerine (GLY), and volatile compounds in e-liquids were analysed by High-Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC). 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay was used to determine the effects of e-liquids on transformed human normal liver epithelial cell line (THLE-2) and human pharyngeal carcinoma cell line (Detroit 562). Nicotine amounts were found to be consistent with product labels. and GLY were not only different between brands but also for products within the same brand. THLE-2 cell viability was inversely correlated with e-liquid concentration. However, decreases in cell viability were not correlated with nicotine amount. Interestingly, effects of several samples on Detroit 562 cells were triphasic; decrease in viability at lower doses, cell survival in mid-concentrations and loss of viability in highest doses. The analytical composition of e-liquids differs greatly among products which corresponds to different cellular effects. Viability of cancer cells does not change in a dose-dependent manner, which suggest that cellular differences may play role in the outcome of these products.

Keywords:

Electronic cigarettes electronic liquids, chromatographic analysis, cell viability,

PDF

___

1. Bahl, V., Lin, S., Xu, N., Davis, B., Wang, Y.H. & Talbot, P. 2012. Comparison of electronic cigarette refill fluid cytotoxicity using embryonic and adult models. Reproductive Toxicology, 34(4): 529-537.
2. Bębenek, P.K., Gholap, V., Halquist, M., Sobczak, A. & Kośmider, L. 2022. E-Liquids from Seven European Countries-Warnings Analysis and Freebase Nicotine Content. Toxics, 10(2): 51.
3. Behar, R.Z., Bahl, V., Wang, Y., Lin, S., Xu, N., Davis, B. & Talbot, P. 2012. A method for rapid dose-response screening of environmental chemicals using human embryonic stem cells. Journal of Pharmacological and Toxicological Methods, 66(3): 238-245.
4. Belushkin, M., Djoko, D.T., Esposito, M., Korneliou, A., Jeannet, C., Lazzerini, M. & Jaccard, G. 2019. Selected Harmful and Potentially Harmful Constituents Levels in Commercial e-Cigarettes. Chemical Research in Toxicology, 33(2): 657-668.
5. Berry, K.M., Reynolds, L.M., Collins, J.M., Siegel, M.B., Fetterman, J.L., Hamburg, N.M., Bhatnagar, A., Benjamin, E.J. & Stokes, A. 2019. E-cigarette initiation and associated changes in smoking cessation and reduction: The Population Assessment of Tobacco and Health Study, 2013-2015. Tobacco Control, 28(1): 42-49.
6. Cheng, T. 2014. Chemical evaluation of electronic cigarettes. Tobacco Control, 23(Supplement 2): ii11-ii17.
7. Cobb, N.K., Byron, M.J., Abrams, D.B. & Shields, P.G. 2010. Novel nicotine delivery systems and public health: the rise of the “e-cigarette”. American journal of public health, 100(12): 2340-2342.
8. Collins, L., Glasser, A.M., Abudayyeh, H., Pearson, J.L. & Villanti, A.C. 2018. E-Cigarette Marketing and Communication: How E-Cigarette Companies Market E-Cigarettes and the Public Engages with E-cigarette Information. Nicotine & Tobacco Research, 21(1): 14-24.
9. CORESTA. 2014. No. 60 - Determination of 1,2-Propylene Glycol and Glycerol in Tobacco and Tobacco Products by Gas Chromatography. https://www.coresta.org/determination-12-propylene-glycol-and-glycerol-tobacco-and-tobacco-products-gas-chromatography-29183 (Date accessed: 20 February 2022).
10. Cullen, K.A., Gentzke, A.S., Sawdey, M.D., Chang, J.T., Anic, G.M., Wang, T.W., Creamer, M.L.R., Jamal, A., Ambrose, B.K. & King, B.A. 2019. e-Cigarette Use among Youth in the United States, 2019. JAMA - Journal of the American Medical Association, 322(21): 2095-2103.
11. El Golli, N., Jrad-Lamine, A., Neffati, H., Rahali, D., Dallagi, Y., Dkhili, H., Ba, N., El May, M.V. & El Fazaa, S. 2016. Impact of e-cigarette refill liquid with or without nicotine on liver function in adult rats. Toxicology Mechanisms and Methods, 26(6): 419-426.
12. Espinoza-Derout, J., Shao, X.M., Bankole, E., Hasan, K.M., Mtume, N., Liu, Y., Sinha-Hikim, A.P. & Friedman, T.C. 2019. Hepatic DNA Damage Induced by Electronic Cigarette Exposure Is Associated With the Modulation of NAD+/PARP1/SIRT1 Axis. Frontiers in Endocrinology, 10(JUN): 320.
13. Esteve, J.M. & Knecht, E. 2011. Mechanisms of autophagy and apoptosis: Recent developments in breast cancer cells. World Journal of Biological Chemistry, 2(10): 232-238.
14. Etter, J.-F., Bullen, C., Flouris, A.D., Laugesen, M. & Eissenberg, T. 2011. Electronic nicotine delivery systems: a research agenda. Tobacco Control, 20(3): 243-248.
15. Farsalinos, K.E., Romagna, G., Allifranchini, E., Ripamonti, E., Bocchietto, E., Todeschi, S., Tsiapras, D., Kyrzopoulos, S. & Voudris, V. 2013. Comparison of the Cytotoxic Potential of Cigarette Smoke and Electronic Cigarette Vapour Extract on Cultured Myocardial Cells. International Journal of Environmental Research and Public Health, 10(10): 5146-5162.
16. Farsalinos, K.E. & Polosa, R. 2014. Safety evaluation and risk assessment of electronic cigarettes as tobacco cigarette substitutes: A systematic review. Therapeutic Advances in Drug Safety, 5(2): 67-86.
17. Giovacchini, C.X., Crotty Alexander, L.E. & Que, L.G. 2022. Electronic Cigarettes: A Pro-Con Review of the Current Literature. The Journal of Allergy and Clinical Immunology, In Practice: S2213-2198(22)00708-5.
18. Harrell, P.T., Naqvi, S.M.H., Plunk, A.D., Ji, M. & Martins, S.S. 2017. Patterns of youth tobacco and polytobacco usage: The shift to alternative tobacco products. American Journal of Drug and Alcohol Abuse, 43(6): 694-702.
19. Hippert, M.M., O’Toole, P.S. & Thorburn, A. 2006. Autophagy in cancer: good, bad, or both? Cancer Research, 66(19): 9349-9351.
20. Holt, A.K., Poklis, J.L. & Peace, M.R. 2021. A Retrospective Analysis of Chemical Constituents in Regulated and Unregulated E-Cigarette Liquids. Frontiers in Chemistry, 9: 752342.
21. Huang, S.J., Xu, Y.M. & Lau, A.T.Y. 2018. Electronic cigarette: A recent update of its toxic effects on humans. Journal of Cellular Physiology, 233(6): 4466-4478.
22. Lee, H.-W., Park, S.-H., Weng, M.-W., Wang, H.-T., Huang, W.C., Lepor, H., Wu, X.-R., Chen, L.-C. & Tang, M.-S. 2018. E-cigarette smoke damages DNA and reduces repair activity in mouse lung, heart, and bladder as well as in human lung and bladder cells. Proceedings of the National Academy of Sciences of the United States of America, 115(7): E1560-E1569.
23. Lerner, C.A., Sundar, I.K., Yao, H., Gerloff, J., Ossip, D.J., McIntosh, S., Robinson, R. & Rahman, I. 2015. 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(2): e0116732.
24. Liebmann, J.E., Cook, J.A., Lipschultz, C., Teague, D., Fisher, J. & Mitchell, J.B. 1993. Cytotoxic studies of paclitaxel (Taxol) in human tumour cell lines. British Journal of Cancer, 68(6): 1104-1109.
25. Madison, M.C., Landers, C.T., Gu, B.-H., Chang, C.-Y., Tung, H.-Y., You, R., Hong, M.J., Baghaei, N., Song, L.-Z., Porter, P., Putluri, N., Salas, R., Gilbert, B.E., Levental, I., Campen, M.J., Corry, D.B. & Kheradmand, F. 2019. Electronic cigarettes disrupt lung lipid homeostasis and innate immunity independent of nicotine. The Journal of Clinical Investigation, 129(10): 4290-4304.
26. Mathis, C., Poussin, C., Weisensee, D., Gebel, S., Hengstermann, A., Sewer, A., Belcastro, V., Xiang, Y., Ansari, S., Wagner, S., Hoeng, J. & Peitsch, M.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 and Molecular Physiology, 304(7): L489-503.
27. Muthumalage, T., Prinz, M., Ansah, K.O., Gerloff, J., Sundar, I.K. & Rahman, I. 2017. Inflammatory and Oxidative Responses Induced by Exposure to Commonly Used e-Cigarette Flavoring Chemicals and Flavored e-Liquids without Nicotine. Frontiers in Physiology, 8: 1130.
28. Romagna, G., Allifranchini, E., Bocchietto, E., Todeschi, S., Esposito, M. & Farsalinos, K.E. 2013. Cytotoxicity evaluation of electronic cigarette vapor extract on cultured mammalian fibroblasts (ClearStream-LIFE): comparison with tobacco cigarette smoke extract. Inhalation toxicology, 25(6): 354-361.
29. Scheffler, S., Dieken, H., Krischenowski, O. & Aufderheide, M. 2015. Cytotoxic evaluation of e-liquid aerosol using different lung-derived cell models. International Journal of Environmental Research and Public Health, 12(10): 12466-12474.
30. Sebaugh, J.L. 2011. Guidelines for accurate EC50/IC50 estimation. Pharmaceutical Statistics, 10(2): 128-134.
31. Shivalingappa, P.C., Hole, R., Van Westphal, C. & Vij, N. 2016. Airway exposure to e-cigarette vapors impairs autophagy and induces aggresome formation. Antioxidants and Redox Signaling, 24(4): 186-204.
32. Sood, A.K., Kesic, M.J. & Hernandez, M.L. 2018. Electronic cigarettes: One size does not fit all. The Journal of Allergy and Clinical Immunology, 141(6): 1973-1982.
33. Tarasenko, Y., Ciobanu, A., Fayokun, R., Lebedeva, E., Commar, A. & Mauer-Stender, K. 2022. Electronic cigarette use among adolescents in 17 European study sites: findings from the Global Youth Tobacco Survey. European Journal of Public Health, 32(1): 126-132.
34. Torres, K. & Horwitz, S.B. 1998. Mechanisms of Taxol-induced cell death are concentration dependent. Cancer Research, 58(16): 3620-3626.
35. Wu, Q., Jiang, D., Minor, M. & Chu, H.W. 2014. Electronic Cigarette Liquid Increases Inflammation and Virus Infection in Primary Human Airway Epithelial Cells. PLOS ONE, 9(9): e108342.
36. Zagoriti, Z., El Mubarak, M.A., Farsalinos, K. & Topouzis, S. 2020. Effects of Exposure to Tobacco Cigarette, Electronic Cigarette and Heated Tobacco Product on Adipocyte Survival and Differentiation In Vitro. Toxics, 8(1): E9.
37. Zahedi, A., Phandthong, R., Chaili, A., Leung, S., Omaiye, E. & Talbot, P. 2019. Mitochondrial Stress Response in Neural Stem Cells Exposed to Electronic Cigarettes. iScience, 16: 250-269.
38. Zhao, J., Zhang, Y., Sisler, J.D., Shaffer, J., Leonard, S.S., Morris, A.M., Qian, Y., Bello, D. & Demokritou, P. 2018. Assessment of reactive oxygen species generated by electronic cigarettes using acellular and cellular approaches. Journal of Hazardous Materials, 344: 549-557.