Electronic cigarettes (e-cigarettes) are an electronic nicotine delivery system that aerosolise liquid typically containing propylene glycol, glycerol, and flavouring agents, with or without nicotine. E-cigarettes have gained popularity as a safer alternative to cigarettes; however, a growing number of studies have shown the harmful effects of e-cigarettes on a range of cellular processes. Given the widespread popularity among young people and the novelty of e-cigarettes, a greater understanding of the biological effects of e-cigarette exposure is needed to further understand their overall health effects. In this study, the researchers investigated the effects of e-cigarette liquid with or without nicotine on V79 lung fibroblast cells. They examined mutagenicity by HPRT (hypoxanthine-guanine-phosphoribosyl-transferase) assay, genotoxicity by comet assay, and the effect on metabolic communication by a metabolic cooperation assay. Moreover, they performed a comprehensive proteome analysis via parallel accumulation serial fragmentation (PASEF) and analysed the data using PEAKS Xpro. They found that e-cigarette liquid displayed no mutagenic or genotoxic effect at the concentration used (dilution of 1.38% corresponding to nicotine concentration of 250 mg), however they observed it did negatively affect metabolic cooperation between V79 cells. Changes in proteome profiles were observed when comparing untreated cells to those treated with e-cigarette liquid without nicotine (ECL) and e-cigarette liquid with nicotine (ECL-N). Specifically, a decrease in the number of proteins detected in both ECL (15%) and ECL-N (23%) was observed when compared to the control group, suggesting a shutdown of protein products. Mitochondrial proteins were found to be the most significantly depleted in both ECL and ECL-N, whereas focal adhesion proteins were found to be upregulated suggesting that ECL liquids can result in mitochondrial dysfunction and increased capacity for migration in V79 cells, both characteristics of many cancer types. Post-translation modification (PTM) profiling revealed significant changes in PTMs following treatment with e-cigarette liquids including the presence of 4-hydroxynonenal (4-HNE), a marker of oxidative stress, in both ECL and ECL-N, but not in the control cells. Additionally, they detected organic biohazardous modifications, such as tri nitro benzene, a highly toxic compound, in ECL and ECL-N treated cells. Taken together, their results show that e-cigarette liquids affect metabolic cooperation in V79 cells and alter proteomic expression profiles that affect global protein synthesis, mitochondrial function, and focal adhesion. These findings highlight the need for more research into the effects of e-cigarettes to fully understand their biological and clinical effects and to ensure the safety of those using this smoking alternative.
How was PEAKS used?
V79 proteins were identified using PEAKS Xpro searched against a UniProtKB database of Cricetulus griseus species (taxon ID 10,029, 56,575 sequences, accessed 22/08/2021) and contamination database as common Repository of Adventitious Protein entries (http://www.thegpm.org/, 116 sequences, accessed 18/10/2019). Identified V79 proteins were selected with false discovery rate (FDR) of less than 0.5% and when at least two unique peptides were detected. Label free quantification (LFQ) of the identified proteins was obtained using PEAKS Q. Relative post translational modification (PTM) profiling of proteins in 4 groups with one sample per group was performed against the built-in 313 post-translational and chemical modifications. For LFQ, the cut off filter criteria for defining confident differentially regulated proteins was a significance score of 20 (−10logP, p < 0.01), two-fold change in log2 up/down ratio, and at least one used peptide affected in any of the 3 treatments in respect to the control group.
Trifunovic, Sara, et al. “Electronic cigarette liquids impair metabolic cooperation and alter proteomic profiles in V79 cells.” Respiratory Research 23.1 (2022): 1-16. doi:10.1186/s12931-022-02102-w
Although still considered a safer alternative to classical cigarettes, growing body of work points to harmful effects of electronic cigarettes (e-cigarettes) affecting a range of cellular processes. The biological effect of e-cigarettes needs to be investigated in more detail considering their widespread use.
In this study, we treated V79 lung fibroblasts with sub-cytotoxic concentration of e-cigarette liquids, with and without nicotine. Mutagenicity was evaluated by HPRT assay, genotoxicity by comet assay and the effect on cellular communication by metabolic cooperation assay. Additionally, comprehensive proteome analysis was performed via high resolution, parallel accumulation serial fragmentation-PASEF mass spectrometry.
E-cigarette liquid concentration used in this study showed no mutagenic or genotoxic effect, however it negatively impacted metabolic cooperation between V79 cells. Both e-cigarette liquids induced significant depletion in total number of proteins and impairment of mitochondrial function in treated cells. The focal adhesion proteins were upregulated, which is in accordance with the results of metabolic cooperation assay. Increased presence of posttranslational modifications (PTMs), including carbonylation and direct oxidative modifications, was observed. Data are available via ProteomeXchange with identifier PXD032071.
Our study revealed impairment of metabolic cooperation as well as significant proteome and PTMs alterations in V79 cells treated with e-cigarette liquid warranting future studies on e-cigarettes health impact.