Pulmonary toxicity and inflammatory response of e-cigarettes containing medium-chain triglyceride oil and vitamin E acetate: Implications in the pathogenesis of EVALI but independent of SARS-COV-2 COVID-19 related proteins

  1. Thivanka Muthumalage
  2. Joseph H. Lucas
  3. Qixin Wang
  4. Thomas Lamb
  5. Matthew D. McGraw
  6. Irfan Rahman

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Abstract

Recently, there has been an outbreak associated with the use of e-cigarette or vaping products, associated lung injury (EVALI). The primary components of vaping products, vitamin E acetate (VEA) and medium-chain triglycerides (MCT) may be responsible for acute lung toxicity. Currently, little information is available on the physiological and biological effects of exposure to these products. We hypothesized that these e-cig cartridges and their constituents (VEA and MCT) induce pulmonary toxicity, mediated by oxidative damage and inflammatory responses, leading to acute lung injury. We studied the potential mechanisms of cartridge aerosol induced inflammatory response by evaluating the generation of reactive oxygen species by MCT, VEA, and cartridges, and their effects on the inflammatory state of pulmonary epithelium and immune cells both in vitro and in vivo. Cells exposed to these aerosols generated reactive oxygen species, caused cytotoxicity, induced epithelial barrier dysfunction, and elicited an inflammatory response. Using a murine model, the parameters of acute toxicity to aerosol inhalation were assessed. Infiltration of neutrophils and lymphocytes was accompanied by significant increases in IL-6, eotaxin, and G-CSF in the bronchoalveolar lavage fluid (BALF). In mouse plasma, eicosanoid inflammatory mediators, leukotrienes, were significantly increased. Plasma from e-cig users also showed increased levels of hydroxyeicosatetraenoic acid (HETEs) and various eicosanoids. Exposure to e-cig cartridge aerosols showed the most significant effects and toxicity compared to MCT and VEA. In addition, we determined at SARS-COV-2 related proteins and found no impact associated with aerosol exposures from these tested cartridges. Overall, this study demonstrates acute exposure to specific e-cig cartridges induces in vitro cytotoxicity, barrier dysfunction, and inflammation and in vivo mouse exposure induces acute inflammation with elevated pro-inflammatory markers in the pathogenesis of EVALI.

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    SciScore for 10.1101/2020.06.14.151381: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIACUC: 2.2 Ethics statement: Institutional biosafety and animal protocol approval: Experiments in this study were performed according to the standards and guidelines approved by The University of Rochester Institutional Biosafety Committee (Study approval #Rahman/102054/09-167/07-186; identification code: 07-186; date of approval: 01/05/2019 and 02/03/2020).
    IRB: Human plasma samples used for lipidomics analysis were from the study conducted at the University of Rochester Medical Center (Rochester, NY, USA) (Institutional Review Board approval RSRB00064337) [10].
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variable2.15 In vivo mouse exposures: Approximately four month old male and female mice with C57BL/6 background were exposed to medium-chain triglyceride oil (MCT)
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The membrane was probed with SP-A antibody (ab115791), ACE2 antibody (ab108252), Furin (ab183495), and TMPRSS2 (ab92323).
    SP-A
    suggested: None
    ACE2
    suggested: None
    ACE2 antibody ( ab108252)
    suggested: None
    Furin ( ab183495)
    suggested: None
    TMPRSS2 ( ab92323)
    suggested: None
    TMPRSS2
    suggested: (Abcam Cat# ab92323, RRID:AB_10585592)
    Experimental Models: Cell Lines
    SentencesResources
    2.10 Cell Culture: Bronchial epithelial cells, BEAS-2B, were cultured in DMEM F-12 50/50 base media and supplemented with 5% FBS, 1% Pen/Strep, and HEPES.
    BEAS-2B
    suggested: None
    At 80% confluency, MM6 cells were serum-deprived in 1% FBS 12-hours before treatment.
    MM6
    suggested: RRID:CVCL_M480)
    Experimental Models: Organisms/Strains
    SentencesResources
    H2-DCF-DA [5 mM] with NaOH [0.01N] was reacted for 30 minutes to prepare the dye.
    H2-DCF-DA
    suggested: None
    2.15 In vivo mouse exposures: Approximately four month old male and female mice with C57BL/6 background were exposed to medium-chain triglyceride oil (MCT)
    C57BL/6
    suggested: None
    Software and Algorithms
    SentencesResources
    2.16 Mouse arterial oxygen saturation: Immediately prior to mouse euthanasia, arterial oxygen saturation was measured in mice by MouseOX plus device (STARR life science, PA).
    STARR
    suggested: (Starr, RRID:SCR_001071)
    2.23 Statistical Analysis: Statistical analysis of data was done by One-Way ANOVA with Tukey’s multiple comparison test for multiple sample groups with one variable and by Two-Way ANOVA with Tukey’s multiple comparison test for multiple sample groups with two variables using GraphPad Prism 8.0.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 14. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.06.14.151381v1 on bioRxiv