Table 2.
Biomarkers of systemic toxicity in ENDS/e-cig exposure
| Sample Matrix (Metabolite/Biomarker) | Methods Used for Analysis of Biomarkers | Groups (n/Group) | Results | Summary of Key Findings | Citation |
|---|---|---|---|---|---|
| Inflammatory biomarkers: cytokines and chemokines | |||||
| IL-1β | |||||
| Saliva | ELISA | Nonsmokers (n = 26); E-cig users (n = 22). |
N/A | Significantly higher levels in e-cig users vs. nonsmokers. | (6) |
| BALF | V-PLEX Plus Proinflammatory Combo 10 Panel (Meso Scale Discovery) | Never-smokers (n = 42); E-cig users (n = 15); Smokers (n = 16). |
Means ± SD = 0.82 ± 0.39 pg/mL Means ± SD = 1.51 ± 1.48 pg/mL Means ± SD = 6.08 ± 5.37 pg/mL |
Significantly higher levels in e-cig users vs. never-smokers.Significantly higher levels in smokers vs. e-cig users. | (7) |
| BALF | Bead-based ELISA (Meso Scale Discovery) | Never-smokers (n = 12); E-cig users (n = 15); Smokers (n = 16). |
N/A | Significantly higher levels in smokers vs. both e-cig users and nonsmokers. | (7) |
| IL-6 | |||||
| Plasma | XL Cytokine Discovery Magnetic Luminex Panel (R&D Systems) | Nonsmokers (n = 26); E-cig users (n = 22). |
N/A | Significantly higher levels in e-cig users vs. nonsmokers. | (6) |
| BALF | V-PLEX Plus Proinflammatory Combo 10 Panel (Meso Scale Discovery) | Never-smokers (n = 42); E-cig users (n = 15); Smokers (n = 16). |
Means ± SD = 0.99 ± 0.56 pg/mL Means ± SD = 1.56 ± 1.02 pg/mL Means ± SD = 4.21 ± 4.93 pg/mL |
Significantly higher levels in e-cig users vs. never-smokers. | (7) |
| Serum | Luminex (magnetic bead kit) | Nonsmokers (n = 5); E-cig users (n = 2); Smokers (n = 5). |
N/A | Significantly higher levels in e-cig users and smokers 3 h after vaping or smoking, respectively, relative to prior levels. | (38) |
| Alveolar macrophages | ELISA (Biotechne) | Never-smokers (n = 8) | N/A | Significantly higher levels after treatment with 0.5% ECVC (with or without nicotine) vs. controls. | (39) |
| IL-8 (CXCL8) | |||||
| Plasma | XL Cytokine Discovery Magnetic Luminex Panel (R&D Systems) | Nonsmokers (n = 26); E-cig users (n = 22). |
N/A | Significantly higher levels in e-cig users vs. nonsmokers. | (6) |
| Alveolar macrophages | ELISA (Biotechne) | Never-smokers (n = 8) | N/A | Significantly higher levels after treatment with 0.5% ECVC (with or without nicotine) vs. controls. | (39) |
| Neutrophils | ELISA (R&D Systems) | Never-smokers (n = 10) | N/A | Significantly higher levels after 6-h exposure to 0.003% ECVE vs. controls. | (40) |
| IL-13 | |||||
| Plasma | XL Cytokine Discovery Magnetic Luminex Panel (R&D Systems) | Nonsmokers (n = 26); E-cig users (n = 22). |
N/A | Significantly higher levels in e-cig users vs. nonsmokers. | (6) |
| ICAM-1 | |||||
| Serum | Luminex (magnetic bead kit) | Nonsmokers (n = 5); E-cig users (n = 2); Smokers (n = 5). |
N/A | Significantly higher levels in e-cig users and smokers 3 h after vaping or smoking relative to prior levels. | (38) |
| MCP-1 | |||||
| Serum | Luminex (magnetic bead kit) | Nonsmokers (n = 5); E-cig users (n = 2); Smokers (n = 5). |
N/A | Significantly higher levels after treatment with 0.05% ECVC (with nicotine) vs. controls. | (38) |
| Alveolar macrophages | ELISA (Biotechne) | Never-smokers (n = 8) | N/A | Significantly higher after treatment with 0.5% ECVC vs. control. | (39) |
| MCSF | |||||
| Serum | Luminex (magnetic bead kit) | Nonsmokers (n = 5); E-cig users (n = 2); Smokers (n = 5). |
N/A | Significantly higher levels in e-cig users and smokers 3 h after vaping or smoking relative to prior levels. | (38) |
| Inflammatory proteases | |||||
| MMP-2 | |||||
| BALF | Western blot normalized to albumin | Nonsmokers (n = 14); E-cig users (n = 14); Smokers (n = 14). |
N/A | Significantly higher levels in both e-cig users and smokers vs. nonsmokers. | (2) |
| MMP-9 | |||||
| Plasma | Custom 9-plex Magnetic Luminex Assay (R&D Systems) | Nonsmokers (n = 26); E-cig users (n = 22). |
N/A | Significantly higher levels in e-cig users vs. nonsmokers. | (6) |
| BALF | Western blot normalized to albumin | Nonsmokers (n = 14); E-cig users (n = 14); Smokers (n = 14). |
N/A | Significantly higher levels in both e-cig users and smokers vs. nonsmokers.No difference between e-cig users and smokers. | (2) |
| Alveolar macrophages | ELISA (Biotechne) | Never-smokers (n = 8) | N/A | Significantly higher levels after treatment with 0.5% ECVC (with or without nicotine) vs. controls. | (39) |
| Neutrophils | ELISA (R&D Systems) and western blot | Never-smokers (n = 10) | N/A | Significantly higher levels after 6-h exposure to 0.003% ECVE vs. controls. | (40) |
| IFNγ | |||||
| Urine | XL Cytokine Discovery Magnetic Luminex Panel (R&D Systems) | Nonsmokers (n = 26); E-cig users (n = 22). |
N/A | Significantly higher levels in e-cig users vs. nonsmokers. | (6) |
| BALF | V-PLEX Plus Proinflammatory Combo 10 Panel (Meso Scale Discovery) | Never-smokers (n = 42); E-cig users (n = 15); Smokers (n = 16). |
Means ± SD = 0.91 ± 0.27 pg/mL Means ± SD = 0.74 ± 0.31 pg/mL Means ± SD = 0.65 ± 0.33 pg/mL |
Significantly higher levels in never-smokers vs. e-cig users.Significantly higher levels in e-cig users vs. smokers. | (7) |
| TNFα | |||||
| Alveolar macrophages | ELISA (Biotechne) | Never-smokers (n = 8) | N/A | Significantly higher levels after treatment with 0.5% ECVC (with nicotine) vs. controls. | (39) |
| Neutrophil elastase (NE) | |||||
| BALF | Western blot normalized to albumin | Nonsmokers (n = 14); E-cig users (n = 14); Smokers (n = 14). |
N/A | Significantly higher levels in both e-cig users and smokers vs. nonsmokers. | (2) |
| Neutrophils | Florescence (FLUOstar omega plate reader (BMG Labtech) | Never-smokers (n = 10) | N/A | Significantly higher levels after 6-h exposure to 0.003% ECVE vs. controls. | (40) |
| Apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) | |||||
| BALF | ELISA | Never-smokers (n = 12); E-cig users (n = 15); Smokers (n = 16). |
Med = 11, IQR = 9–15 ng/mL Med = 22, IQR = 14–35 ng/mL Med = 37, IQR = 21–64 ng/mL |
Significantly higher levels in both e-cig users and smokers vs. nonsmokers. | (37) |
| Caspase-1 | |||||
| BALF | ELISA | Never-smokers (n = 12); E-cig users (n = 15); Smokers (n = 16). |
Med = 12, IQR = 6-14 pg/mL Med = 16, IQR = 9-35 pg/mL Med = 42, IQR = 27-68 pg/mL |
Significantly higher levels in smokers vs. both e-cig users and nonsmokers. | (37) |
| Oxidative stress, DNA damage, and lipid peroxidation biomarkers | |||||
| 8-Isoprostane | |||||
| Urine | ELISA | Nonsmokers (n = 26); E-cig users (n = 22). |
N/A | Significantly higher levels in e-cig users vs. nonsmokers. | (6) |
| Spot urine | ELISA (R&D) | Nonsmokers (n = 20); E-cig users (n = 20); Smokers (n = 13). |
Means ± SD = 411.2 ± 287.4 pg/mg creatinine Means ± SD = 750.8 ± 433 pg/mg creatinineMeans ± SD = 784.2 ± 546.1 pg/mg creatinine |
Significantly higher levels in e-cig users vs. nonsmokers.No significant difference between e-cig users and smokers. | (4) |
| 8-OHdG | |||||
| Urine | HT 8-Oxo-dG Human ELISA (Trevigen) | Nonsmokers (n = 26); E-cig users (n = 22). |
N/A | Significantly higher levels in e-cig users vs. nonsmokers. | (6) |
| Spot urine | DNA Damage (8-OHdG) ELISA Kit (Stress Marq Biosciences) | Nonsmokers (n = 20); E-cig users (n = 20); Smokers (n = 13). |
Means ± SD = 221.6 ± 157.8 ng/mg creatinine Means ± SD = 442.8 ± 300.7 ng/mg creatinine Means ± SD = 388 ± 235 ng/mg creatinine |
Significantly higher levels in e-cig users vs. nonsmokers.No significant difference between e-cig users and smokers. | (4) |
| ROS | |||||
| Alveolar macrophages | DCFDA assay (Abcam) | Never-smokers (n = 8) | 50-fold increase in ECVC treated AM (with or without nicotine) vs. untreated controls | Significantly higher levels after treatment with 0.5% ECVC (with or without nicotine) vs. controls. | (39) |
| MDA | |||||
| Plasma | Colorimetric lipid peroxidation assay (Oxford Biomedical Research) | Smokers switch to e-cigs (n = 42); Smokers switch to dual use (n = 24); Smoker controls (no switch; n = 20). |
1.12 ± 0.1 (prior); 1.01 ± 0.1 (1 mo) nmol/L1.28 ± 0.1 (prior); 1.09 ± 0.1 (1 mo) nmol/L1.12 ± 0.3 (prior); 1.15 ± 0.2 (1 mo) nmol/L | Significant decrease in smokers who switched to e-cigs or dual use (cigarettes and e-cigs) for 1 mo relative to BSL. No change in smoker controls. | (20) |
| CC16 | |||||
| Serum | N/A | Occasional smokers (n = 23); Vape session: Sham vape session: | 0.51 (BSL)–0.64 (post) µg/mg creatinine0.63 (BSL)–0.49 (post) µg/mg creatinine | E-cig use significantly increased median levels relative to BSL after 30 min e-cig use vs. sham vaping. | (43) |
| Serum | Latex immunoassay using rabbit anti-CC16 antibody | Smokers (n = 25) Vape session (nicotine): Vape session (no nicotine): Sham vape session: | Δ: +1.2 ± 0.3 µg/L (after vaping-BSL) Δ: +1.1 ± 0.3 µg/L (after vaping-BSL) Δ: −0.5 ± 0.2 µg/L (after vaping-BSL) |
E-cig use significantly increased means ± SE levels relative to BSL after 30 min e-cig use (with or without nicotine) vs. sham vaping. | (44) |
| EVALI-specific biomarkers: vitamin E acetate (VEA) | |||||
| BALF | Isotope dilution mass spectrometry | EVALI patients (n = 51); Nonusers (n = 52); E-cig users (n = 18); Smokers (n = 29) |
present in 48/51;present in 0/52; present in 0/18; present in 0/29 | VEA present in most EVALI samples but not in any e-cig user, smoker or nonuser samples. | (45) |
| Transcriptomic/gene expression biomarkers | |||||
| Bronchial brushings | GeneChip Human Transcriptome Array 2.0 (Affymetrix) | Never-smokers (n = 42); E-cig users (n = 15); Smokers (n = 16). |
181 DETs unique to e-cig users | 2,452 DETs among groups (for 2,093 genes) | (7) |
| Oral cells | RNA-seq | Nonsmokers (n = 27); E-cig users (n = 42); Smokers (n = 24). |
∼50% more DETs than e-cig users | Significant numbers of DETs in both e-cig users and smokers vs. nonsmokers. | (27) |
| SAEAlveolar Macrophages | TruSeq v2 mRNA library prep RNA-sequencing (2 × 125 bp) (Illumina HiSeq. 2500) | Never-smokers (n = 10) given e-cig with nicotine (n = 7) or without nicotine (n = 3). | 71 significantly altered genes vs. BSL; 65 significantly altered genes vs. BSL;27 significantly altered genes vs. BSL; 61 significantly altered genes vs. BSL | For both groups, significantly altered pathways were associated with nicotine receptors and downstream p53 targets.No standard pathways identified in alveolar macrophages. | (59) |
| Primary HBECs | GeneChip Human Gene 1.0 ST Array (Affymetrix) | Nonsmoker (donor; n); E-cig aerosol exposure; Cig smoke exposure; Air (control). | 546 genes significantly differentially expressed among the three groups;493 genes differentially expressed between tobacco and menthol e-cig vapor. | Genes altered in both e-cig and smoke groups associated with cilium assembly/movement, apoptosis, xenobiotic and oxidative stress, and DNA damage pathways.Genes altered uniquely in e-cig group associated with cell division and cell cycle regulation pathways. | (60) |
| Primary NHBEs | RNA-seqResults relative to H2O controls | Diacetyl (2,3-butanedione), 2,3-pentanedione. | 163 differentially expressed genes;568 differentially expressed genes | Differentially expressed genes associated with cytoskeletal- and cilia-related pathways. | (24) |
| Differentiated HBECs | RNA-seq (Illumina)Results relative to air controls | Nonsmokers (donors; n = 2); E-cig vapor exposure; Cig smoke exposure; Air (control). |
57 differentially expressed genes (1 h);49 differentially expressed genes (0 h) | Genes altered uniquely in e-cig group associated with the cell cycle, response to hypoxia, response to organic substances, apoptosis and acute inflammatory response. | (61) |
| Nasal epithelium biopsies | RNA-seqResults relative to nonsmokers | Nonsmokers (n = 13); E-cig users (n = 12); Smokers (n = 14). |
358 genes significantly downregulated;53 genes significantly downregulated | All 53 genes downregulated in smokers were also downregulated in e-cig users. | (62) |
| Exosomal small RNAs as circulating biomarkers | |||||
| Plasma-derived exosomes | RNA-seq (Illumina NextSeq. 500)Results relative to non-smokers | Non-smokers (n = 8); E-cig users (n = 7);Cigarette smokers (n = 7); Waterpipe smokers (n = 7); Dual smokers (n = 7). |
17 miRNAs, 7 tRNAs and 5 piRNAs differentially expressed in e-cig users | 13 miRNAs were significantly upregulated, and 4 miRNAs were significantly downregulated in e-cig users compared to non-smokers. | (64) |
| DNA methylation biomarkers | |||||
| Bronchial brushings | Infinium Methylation EPIC BeadChip (Illumina) | Never-smokers (n = 10); E-cig users (n = 12); Smokers (n = 10). |
14 unique CpGs differentially expressed | 451 differentially methylated CpGs (273 genes) among the three groups. | (7) |
| Peripheral blood leukocytes | DNA methylation LINE-1 kit (Active motif)RNA-seq Results relative to nonsmokers | Nonsmokers (n = 15); E-cig users (n = 15); Smokers (n = 15). |
18% hypomethylation in LINE1 elements;13% hypomethylation in LINE1 elements | No significant difference in levels of LINE-1 hypomethylation between smokers and e-cig users. | (23) |
| Proteomic biomarkers | |||||
| Bronchial brush biopsies | LC-MS/MSResults relative to nonsmokers | Nonsmokers (n = 8); E-cig users (n = 9); Smokers (n = 9). |
191 differentially expressed proteins;292 differentially expressed proteins | 78 proteins similarly altered in vapers and smokers.131 proteins exclusively altered in vapers.14 uniquely altered pathways in vapers. | (48) |
| Induced sputum | Q-Exactive mass spectrometer coupled to UltiMate 3000 nano HPLC system (Thermo Scientific). | Never-smokers (n = 15); E-cig users (n = 15); Smokers (n = 14). |
5 significantly upregulated proteins;66 significantly upregulated proteins;23 significantly upregulated proteins | 15 proteins commonly upregulated in smokers and vapers.∼81 proteins significantly altered in e-cig users relative to never-smokers, 44 in smokers relative to never-smokers. | (17) |
| MUC5AC | |||||
| Induced sputum | Stable-isotope-labeled mass spectrometry with parallel reaction monitoring analysis | Never-smokers (n = 15); E-cig users (n = 15); Smokers (n = 14). |
Means ± SD = 15 ± 6 pmol/mL; Means ± SD = 58 ± 21 pmol/mL; Means ± SD = 132 ± 58 pmol/mL |
Significantly higher levels in both e-cig users and smokers vs. nonsmokers. | (17) |
BALF, bronchoalveolar lavage fluid; BSL, baseline; CpGs, CpG sites; DETs, differentially expressed transcripts; ECVC, e-cigarette vapor condensate; ECVE, e-cig vapor extract; ELISA, enzyme-linked immunosorbent assay; EVALI, e-cigarette or vaping-associated lung disease; HBECs, human bronchial epithelial cells; IQR, interquartile range; LC-NSI-HRMS/MS, liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry; med, median; N/A, details not available; NHBEs, normal human bronchial epithelial cells; SAE, small airway epithelium; SD, standard deviation.