Cytotoxic and genotoxic effects of e-liquids and their potential associations with nicotine, menthol and phthalate esters.


Environmental Health Program, Saudi Arabia. Electronic address: [Email]


In this study, we determined DNA damage and chromosome breakage (indicators of genotoxicity) and cell viability (an indicator of cytotoxicity) in human lymphoblastoid TK6 and Chinese hamster ovary (CHO) cells treated with 33 e-liquids using in vitro single cell gel (comet), micronucleus (MN), and trypan blue assays, respectively. We also measured the contents of nicotine, five phthalate esters, and DL-menthol in the e-liquids to examine their effects on DNA damage, chromosome breakage, and cell viability. Our chemical analyses showed that: (1) six e-liquids had nicotine ≥2-fold higher than the manufacture's label claim (2-3.5 mg); (2) both dimethyl- and dibutyl-phthalate levels were >0.1 μg/g, i.e., their threshold limits as additives in cosmetics; and (3) the DL-menthol contents ranged from 0.0003 to 85757.2 μg/g, with those of two e-liquids being >1 mg/g, the threshold limit for trigging sensory irritation. Though all the e-liquids induced DNA damage in TK6 cells, 20 resulted in cell viabilities ≤75%, indicating cytotoxicity, yet the inverse relationship between cell viability and DNA damage (r = -0.628, p = 0.003) might reflect their role as pro-apoptotic and DNA damage inducers. Fifteen e-liquids induced MN% in TK6 cells ≥3-fold that of untreated cells. Some of the increase in %MN might be false due to high cytotoxicity, yet six brands showed acceptable cell viabilities (59-71%), indicating chromosome damage. DNA damage and %MN increased when the TK6 cells were exposed to metabolic activation. The CHO cells were less sensitive to the genotoxic effects of the e-liquids than the TK6 cells. DL-menthol was found to be associated with decreased cell viability and increased DNA damage, even at low levels. We cannot dismiss the presence of other ingredients in e-liquids with cytotoxic/genotoxic properties since out of the 63 different flavors, 47 induced DNA damage (≥3-folds), and 26 reduced cell viability (≤75%) in TK6 cells.


Cell viability,Comet assay,Menthol,Micronucleus,Nicotine,Phthalate esters,e-cigarettes,

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