The volatile compounds (VOCs) disclosed by the GC/MS analysis on the e-cig aerosol were in agreement with the literature3, 5, 6. The main VOCs detected in our study were propylene glycol (PG), nicotine and vegetable glycerin (VG), together with other minor compounds and flavours (such as 1,2-propanediamine, methyl propionate, indole, propanoic acid 1-methylpropyl ester, acetol, 1-methoxy-2-propyl acetate, 3-hexen-1-ol, diacetyl and acrolein) (Table 1). In particular, heating of VG produces temperature-dependent amounts of hazardous aldehydes (such as formaldehyde, acetaldehyde and acrolein), due to thermal decomposition by free-radical dehydration of glycerol: formaldehyde, acetaldehyde and acrolein are formed at 600 °C, whereas acrolein is produced in some ionic environments at 350 °C7.

Table 1 Volatile compounds (VOCs) detected in the first and last treatment chambers during exposure to e-cig vapour. Full size table

Since variables such as device brand, device wattage, resistive heating wire, nicotine concentration, PG/VG ratio and puff duration could significantly influence the emission of hazardous compounds (e.g. acrolein) and the rate at which nicotine is emitted per unit time8, we recorded the changes in the VOC profile throughout the exposure to e-cig vapour. No significant differences (P > 0.05) were found in the VOC composition of the different exposure chambers during animal treatment. This finding and the constant PG/VG ratio confirm that the same e-cig aerosol composition was supplied to the animals and no devices overheated. On the other hand, no trace of formaldehyde was detected, probably due to the procedure used to determine the VOC profile that entails derivatization steps, leading to selective determination of VOCs. However, no significant changes (P > 0.05) were detected in the amounts of acrolein (0.02–0.03% of total VOCs), VG or PG, suggesting that similar amounts of other aldehydes (not detected) were supplied throughout the treatment cycles, so the effects detected on the rats are related to all the compounds formed by e-liquid vaporization and present in the aerosol.

To explore whether e-cigs induce toxicological effects, such as those involving cytochrome P450 (CYP) changes9, we analysed the modulation of carcinogen-metabolizing enzymes in the lungs of rats exposed to e-cig vapour (see Methods). We observed a significant increase in CYP1A1/2 (activating, for example, polychlorinated biphenyls, aromatic amines, dioxins and PAHs), CYP2B1/2 (activating olefins and halogenated hydrocarbons), 2C11 (activating nitrosamines and mycotoxins) and CYP3A (activating hexamethyl phosphoramide and nitrosamines) documented by the sharp rise in the corresponding probes (Fig. 1a). Extrapolated to humans, the corresponding boosted CYP-linked monooxygenases would predispose a subject to an enhanced cancer risk from the widely bioactivated e-cig vapour procarcinogens associated with an increased risk of lung cancer with CYP induction and/or CYP polymorphisms10. The overproduction of reactive oxygen species (ROS) resulting from CYP induction is one of the well-documented ways in which CYP can play a key role in new cancer occurrence via a co-carcinogenesis mechanism11. Until the early 1990s, this phenomenon was initially associated only with induction of the CYP2E1 isoform. Then in 1996, we found that virtually all upregulated CYP isoforms can overproduce ROS12 primarily by uncoupling the CYP catalytic cycle.

Figure 1 Metabolic/antioxidant enzymes and free radical yield in e-cig-exposed rat lung. (a) Cytochrome P450 (CYPs) is a superfamily of major isoenzymes involved in drug metabolism. CYP activities lead to the bioactivation of ubiquitous pre-mutagens and pre-carcinogens as well as ROS generation linked to their catalytic cycle. Data were obtained through enzymatic assays performed on microsomal lung fractions using several specific probes: MROD (CYP1A2-like) increased up to 262%, PROD (2B1/2) 384%, APD (3A, 1A, 2A, 2D) 19% P < 0.05, 16-α TOH (2B1/2C11) 48% (P < 0.01), 17-TOH (3A1) 41% (P < 0.01). (b) EPR spectra of nitroxide radicals observed in rat lung tissues in control samples (green spectra), and in e-cig vapour-treated samples (red spectra). (c) EPR intensity of the first spectral line of the observed nitroxide radicals (arbitrary units). (d) Antioxidant enzymes: CAT, NQO1 and SOD were reduced more than 32% (P < 0.01). (e) Transferases shown here are involved in the detoxifying step of xenobiotic metabolism making drugs or toxins more water-soluble. They also contribute to preserving DNA from adduct formation converting carcinogens into inactive or less toxic compounds: UDP-GT unchanged, GST 28% loss (P < 0.01). Each bar represents the means ± S.D. of ten measurements performed on ten rats, * P < 0.05, ** P < 0.01, two-tailed t-test. Full size image

We then used the electron paramagnetic resonance “EPR-radical probe” technique to evaluate the free radical content in lung. We found a significant increase in radical species yield in the lung (Fig. 1b,c). Our data are consistent with those recently reported by Sussan et al.13 showing e-cig vapour induced the development of oxidative stress in the lung. It is reasonable to hypothesize that the CYP induction found here, together with the free radicals present in the aerosol13, 14, contributed to the higher levels of ROS detected in exposed rats. Notably, we observed that the antioxidant enzymes catalase, DT-diaphorase and superoxide dismutase were all markedly reduced (Fig. 1d). Conversely, the conjugating phase II glutathione S-transferases, mainly involved in xenobiotic detoxification, were noticeable decreased, whereas UDP-glucuronyl-transferase was substantially unchanged (Fig. 1e). Thus, the reduced activity of antioxidant machinery, the free radicals reported to be present in vapour along with those generated by CYP induction found here can contribute to the inflammatory response6, 13, 15 and suggest an impairment of redox homeostasis in the lung.

To examine whether these phenomena affect the antioxidant power, we measured the systemic antioxidant capacity using the ferric reducing antioxidant power (FRAP) approach, finding a markedly reduced FRAP value in the lung (Fig. 2a). A similar trend was observed in plasma, even if statistical significance was not reached (P = 0.059) (Fig. 2b). Interestingly, plasma FRAP levels and measurement of carbonyl residues (CO) as biomarkers of oxidative injury to proteins were inversely correlated in e-cig vapour-exposed rats (Fig. 2c). In contrast, animals from the control group showed the opposite behaviour (Fig. 2d), indicating that control animals were able to increase their antioxidant capacity in relation to oxidative stress, while the lack of a protective antioxidant response in e-cig-exposed animals might explain the reduced FRAP level associated with CO formation. We also measured guanosine oxidation to 8-hydroxy-2′-deoxyguanosine (8-OHdG). 8-OHdG is one of the most extensively studied and abundant free radical-induced oxidative DNA lesions, which also correlates with mutagenesis in bacterial and mammalian cells16. Based on this evidence, 8-OHdG has been widely used as a biomarker to evaluate the load of oxidative stress and carcinogenesis17. We found that 8-OHdG markedly increased in the lungs of e-cig rats (Fig. 2e). This was supported by an inverse correlation between FRAP and 8-OHdG in lung tissue from exposed animals (Fig. 2f).

Figure 2 Systemic antioxidant capacity, oxidative DNA damage and lipidomics. (a) FRAP lung, loss >33% (P < 0.05). (b) FRAP plasma (P = 0.059). (c) FRAP plasma from e-cig group inversely correlated vs CO (r = 0.930, P < 0.001). (d) FRAP plasma from control group positively correlated vs CO (r = 0.880, P < 0.01). (e) 8-OHdG lung levels markedly increased ~288% (P < 0.01). (f) FRAP lung from e-cig group inversely correlated vs 8-OHdG (r = 0.845, P < 0.05). Data (n = 5 measurements per group) are expressed as means ± standard error of the mean (SEM), analysed by one-way analysis of variance (ANOVA) (g) left side, content of esterified cholesterol, total cholesterol and triglycerides (mg/dL) determined by GC/MS and GC/FID for qualitative and quantitative analysis, respectively, on Control and E-cig groups. Right side, sum of C18:1 trans isomers, saturated fatty acids (SFA), polyunsaturated fatty acids (PUFA) and PUFA n-6 series (PUFA n-6) in percentage (%) of total fatty acids determined by GC/FID on Control and E-cig groups. Each bar represents the means ± S.D. of ten measurements performed on ten * P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed t-test. Full size image

Insights into the redox imbalance also emerged from the study of the lipidome (Fig. 2g). The main lipid classes (free fatty acids, free cholesterol, esterified cholesterol and triglycerides) were determined by GC/MS. After e-cig aerosol exposure, the overall lipid composition of rat plasma was markedly affected with significant increases in the content of esterified cholesterol (EC), total cholesterol (TC) and triglycerides (TG) (P < 0.05) (Fig. 2g). These results agree with those reported by recent literature studies18 showing increased concentrations of TG, VLDL and TG/HDL ratios after four weeks’ intraperitoneal injection of nicotine and e-cig refill liquid containing nicotine in rats. Since the liver is the main organ responsible for cholesterol and lipoprotein synthesis, exposure to e-cig vapours might have affected rat liver function. On the other hand, the nicotine could have triggered the release of catecholamines and cortisol, in turn leading to activation of adenyl cyclase in adipose tissue and lipolysis of stored TG, with a subsequent increase in plasma VLDL and TG19. Tobacco smoking is known to lead to significantly higher serum concentrations of cholesterol and TG20,21,22. We did not explore the mechanism underlying this phenomenon, but nonetheless consider the finding noteworthy. In addition, we detected significant variations in the fatty acid composition of plasma: in particular, the sum of C18:1-trans isomers significantly (P < 0.001) increased probably due to the interaction of reactive VOCs generated by e-cig and plasma lipids. A significant increase in saturated fatty acids (SFA) was also found, whereas the content of polyunsaturated fatty acids (PUFA) and PUFA n-6 series noticeably decreased (Fig. 2g). Recently, Shen et al.23 demonstrated that alterations in cellular glycerophospholipid biosynthesis are an important consequence of e-cig vapour exposure due to enriched gene expression, and this could explain the fatty acid differences found here. However, in-depth histopathological investigation of diverse organs (such as liver) could yield more information on the lipidome changes found after e-cig aerosol exposure, and thereby shed more light on the underlying mechanisms involved.

To investigate the putative genotoxic potential of e-cig vapour, we considered various genetic endpoints at chromosomal and gene level in rat peripheral blood and urine, which served as body collectors of mutagenic metabolites. We observed that e-cigs produce extensive DNA damage in leukocytes measured as tail comet length of the fragmented DNA determined by single- and double-strand breaks (Fig. 3a,b). These data are in line with previous in vitro outcomes on HaCaT, UMSCC10B, and HN30 cell lines exposed to nicotine-containing and nicotine-free vapour extracts from two popular e-cig brands24. We also found that e-cig vapour determines an increase in the percentage of immature micronucleated reticulocytes (MN-RET) over normal reticulocyte RT (Fig. 3c,d). These results indicate that the mixture of chemical compounds generated by e-cigs leads to chromosome fragmentation and possibly damage to the mitotic spindle or centromeres. We observed a concomitant severe hematopoietic depression on exposed rats (Fig. 3e). Next, the urine of e-cig-exposed animals induced a dose-dependent increase in the number of S. typhimurium revertants in different strains. The highest sensitivity was shown by the TA100 strain (Fig. 3f), revealing base substitutions, and YG1024 (Fig. 3g), disclosing frame-shift mutations with an increased sensitivity to nitroarenes and aromatic amines. Mutant induction was affected by the S9 external metabolic activation system, suggesting both mutagenic and promutagenic metabolites in the urine.

Figure 3 Genotoxicity of e-cig vapour. (a) Distribution of individual median TI% for the alkaline Comet assay. (b) Box-plot of TI%: primary DNA damage increase (P < 0.001). (c) Representative image of micronuclei (yellow MN-RET, orange RET, green erythrocytes (E). (d,e) MN-RET vs RET and RET vs E plus RET; MN-RET vs RET increased (P < 0.05); hematopoietic depression up to 50% loss, as RT fraction of total red blood cells, (P < 0.001). Error bars ± S.D. * P < 0.05; *** P < 0.001 two-tailed t-test. (f,g) Urinary mutagenesis: TA100 and YG1024 S. typhimurium revertants/plate increased in a dose-dependent manner ± S9 mix. * P < 0.05; ** P < 0.01; *** P < 0.001 Bonferroni’s test). Full size image

Despite its shortcomings, the work presented here strongly raises the possibility that e-cig consumption under certain conditions leads to toxicological outcomes directly and indirectly damaging DNA in the rat. As shown in Table 1, our GC/MS analysis of the vapour was consistent with the literature3 confirming the presence, among others, of acrolein, toxic and mutagenic compounds25. However, our study currently precludes any cause-effect speculation, ascribing responsibility for the effects detected to the vapour as a whole rather than the single components. Our results should be construed as stemming from a preliminary study which was not conceived to replicate human vaping conditions, but to demonstrate if exposure to the chemical cocktail derived from e-cig liquid vaporization can result in toxicological injury.

As these detrimental phenomena are typically induced by conventional cigarettes26,27,28, the erroneous belief that e-cigs are safe should be retracted and suitable measures implemented to protect public health. Our study should be seen as the starting point for further investigations designed to confirm the harmful health impact of e-cigs, and a thorough analysis of their risk-benefit ratio, particularly after long-term exposure and under different usage conditions.