电子烟烟液及其主要化学成分的体外细胞毒性评价
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摘要
为了解电子烟烟液的细胞毒性以及其主要成分对细胞毒性的影响,通过比对乳酸脱氢酶活性实验(LDH)、溴脱氧尿嘧啶核苷掺入实验(BrdU)、WST-1实验和CCK-8实验检测电子烟烟液对中国仓鼠卵巢细胞(CHO细胞)和人肺癌细胞(A549细胞)的细胞毒性敏感程度,建立了适用于电子烟烟液细胞毒性评价方法,并对16种电子烟烟液进行了分析,通过线性回归分析和Pearson相关分析探讨了电子烟烟液中的烟碱、1,2-丙二醇和丙三醇等与电子烟烟液细胞毒性的相关性。结果表明:①不同实验评价电子烟烟液细胞毒性的IC50值大小为CCK-8 细胞毒性最高相差达5.47倍,但是其细胞毒性与烟液中烟碱、1,2-丙二醇和丙三醇含量(质量分数)的线性回归系数及Pearson相关系数均显著低于线性相关所需水平(P <0.05)。因此,CHO细胞和CCK-8实验更适合于电子烟烟液的细胞毒性评价,该实验范围内电子烟烟液的细胞毒性与其主要成分(烟碱、丙三醇和1,2-丙二醇)含量之间无明显线性相关性。
In order to investigate the cytotoxicity of e-cigarette liquids(e-liquids) and the effects of their main chemical components on the cytotoxicity, a method for evaluating the cytotoxicity of e-liquids was established by comparing responses and detection sensitivities using a range of cytotoxicity assays. These included lactate dehydrogenase assay(LDH), bromodeoxyuridine incorporation assay(BrdU), WST-1 assay, and CCK-8 assay for cytotoxicity of e-liquids to Chinese hamster ovary cells(CHO cells) and human lung cancer cells(A549 cells). A total of 16 e-liquids were analyzed to assess the responses from the market collection. Linear regression and Pearson correlation analyses were used to study the correlation between the cytotoxicity of the e-liquids and the contents of nicotine, 1,2-propylene glycol and glycerol in the e-liquids. The results showed that: 1) The IC50 values of the cytotoxicity of the e-liquids were in the sequence of CCK-8 < WST-1 < BrdU
In order to investigate the cytotoxicity of e-cigarette liquids(e-liquids) and the effects of their main chemical components on the cytotoxicity, a method for evaluating the cytotoxicity of e-liquids was established by comparing responses and detection sensitivities using a range of cytotoxicity assays. These included lactate dehydrogenase assay(LDH), bromodeoxyuridine incorporation assay(BrdU), WST-1 assay, and CCK-8 assay for cytotoxicity of e-liquids to Chinese hamster ovary cells(CHO cells) and human lung cancer cells(A549 cells). A total of 16 e-liquids were analyzed to assess the responses from the market collection. Linear regression and Pearson correlation analyses were used to study the correlation between the cytotoxicity of the e-liquids and the contents of nicotine, 1,2-propylene glycol and glycerol in the e-liquids. The results showed that: 1) The IC50 values of the cytotoxicity of the e-liquids were in the sequence of CCK-8 < WST-1 < BrdU
引文
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[2] Bahl V,Lin S,Xu N,et al. Comparison of electronic cigarette refill fluid cytotoxicity using embryonic and adult models[J]. Reproductive Toxicology,2012,34(4):529-537.
[3] Behar R Z,Davis B,Wang Y,et al. Identification of toxicants in cinnamon-flavored electronic cigarette refill fluids[J]. Toxicology in Vitro,2014,28(2):198-208.
[4] Farsalinos K E,Romagna G,Allifranchini E,et al.Comparison of the cytotoxic potential of cigarette smoke and electronic cigarette vapour extract on cultured myocardial cells[J]. International Journal of Environmental Research and Public Health,2013,10(10):5146-5162.
[5] Romagna G,Allifranchini E,Bocchietto E,et al.Cytotoxicity evaluation of electronic cigarette vapor extract on cultured mammalian fibroblasts(ClearStream-LIFE):comparison with tobacco cigarette smoke extract[J]. Inhalation Toxicology,2013,25(6):354-361.
[6]李翔,谢复炜,刘惠民.新型烟草制品毒理学评价研究进展[J].烟草科技,2016,49(1):88-93.LI Xiang,XIE Fuwei,LIU Huimin. Recent advances in toxicological evaluation of novel tobacco products[J].Tobacco Science&Technology,2016,49(1):88-93.
[7]李宁,万文婷,金林红. CCK-8法和MTT法检测人前列腺癌PC3细胞活性的最佳条件比较研究[J].贵州大学学报(自然科学版),2009,26(3):18-20,24.LI Ning, WAN Wenting, JIN Linhong. Optimal experiment conditions of comparison between CCK-8and MTT in cell proliferation assays on PC3 cell[J].Journal of Guizhou University(Natural Sciences),2009,26(3):18-20,24.
[8] Fotakis G,Timbrell J A. In vitro cytotoxicity assays:comparison of LDH,neutral red,MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride[J]. Toxicology Letters,2006,160(2):171-177.
[9] Lappalainen K,J??skel?inen I,Syrj?nen K,et al.Comparison of cell proliferation and toxicity assays using two cationic liposomes[J]. Pharmaceutical Research,1994,11(8):1127-1131.
[10] Vega-Avila E,Pugsley M K. An overview of colorimetric assay methods used to assess survival or proliferation of mammalian cells[J]. Proceedings of the Western Pharmacology Society,2011,54:10-14.
[11] Lerner C A,Sundar I K,Yao H,et al. 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[J].PLoS One,2015,10(2):e0116732.
[12] Cheng T R. Chemical evaluation of electronic cigarettes[J]. Tobacco Control,2014,23(S2):ii11-ii17.