Association of 1 Vaping Session With Cellular Oxidative Stress in Otherwise Healthy Young People With No History of Smoking or Vaping

Abstract

This randomized clinical crossover trial evaluates the association of a single session of electronic cigarette vaping with cellular oxidative stress in healthy young people who do not smoke compared with individuals with long-term tobacco cigarette or electronic cigarette use.

Like tobacco cigarette (TCIG) smoking, long-term electronic cigarette (ECIG) vaping in young people is associated with elevated cellular oxidative stress (COS), which is important in the pathogenesis of many diseases, including atherosclerosis.¹ As with TCIG smoking,² even infrequent ECIG use may be associated with adverse biological effects with implications for future health risks. Importantly, the proportion of high school students who have used ECIGs within 1 month of the time of study has skyrocketed, approaching 30% in the US.^3,4 The purpose of this study was to evaluate the association of a single session of ECIG vaping on COS in immune cells in young people who do not smoke or vape compared with young people with long-term TCIG or ECIG use.

Methods

Among 32 healthy participants in this randomized clinical crossover trial, the mean (range) age was 24 (21-33) years, and 19 individuals were male. Nine individuals had long-term (more than 1 year) TCIG use, 12 had long-term ECIG use, and 11 did not have any history of TCIG or ECIG use. Baseline characteristics did not differ between the 3 groups, and tobacco-use burden at baseline did not differ between the ECIG and TCIG groups (eMethods in Supplement 2).¹ Immune cells were collected before and 4 hours following supervised sessions of either vaping, using an ECIG with 5% nicotine, or sham vaping (puffing on a straw) in random sequence as previously described.⁵ We used established flow cytometry methods¹ to determine COS in immune cells by investigators blinded to the study groups. The protocols were approved by the institutional review board of the University of California, Los Angeles, and registered on ClinicalTrials.gov. All participants gave written informed consent. The Kruskal-Wallis test was used to compare the 3 tobacco use groups and the Mann-Whitney U test was used to compare the vaping vs sham groups. Tests were 2-tailed, and the level of significance was set at P < .05. As this was a small, exploratory study, we did not adjust for multiple comparisons.

Results

Representative data of measures of COS have been published.¹ Consistent with our prior findings,¹ all baseline measures of COS were lowest in individuals who never smoked, intermediate in individuals who currently used ECIGs, and highest in individuals who currently smoked TCIGs (data not shown). Exposure to a single vaping session, but not the sham-control session, increased the percentage of CD45⁺ immune cells that were positive for CellROX Green 1.3-fold compared with the nhistory of smoking or vaping or individuals who currently used ECIGs or TCIGs (Figure 1).

Figure 1. Effect of Exposure to a Single Vaping Session on Measures of Cellular Oxidative Stress (COS) in Total Immune Cells by Group.

Peripheral blood mononuclear cells and flow cytometry were used to determine expression (percentage of positive cells and median fluorescence intensity [MFI]) of fluorochromes that determine cellular oxidative stress (COS). CellROX Green was used for total (cytoplasmic and nuclear) cellular reactive oxygen species content and CellROX Deep Red for cytoplasmic reactive oxygen species content. Measures of COS were determined among groups according to tobacco use at baseline and after a single exposure to electronic cigarette vaping with an electronic cigarette with 5% nicotine (labeled as vaping) or sham vaping (puffing on a straw labeled as sham). Fluorescence intensity of a positive cell population was compared with a negative cell population (fluorescence minus one negative control for staining) (∆MFI). The compared groups were individuals with no history of smoking or vaping, individuals who used electronic cigarettes, and individuals who smoked tobacco cigarettes. The value of each measurement (percentage of positive cells and ∆MFI) after the intervention (sham vs vaping) was normalized by the value of each measurement at baseline within each person and was expressed as fold to the baseline value.

^aP < .01.

Given heterogeneity of cellular responses, we determined the effect of a single vaping session on COS in immune cell subtypes among groups. Compared with sham control, acute vaping increased the percentage of CD14²⁺CD16⁺ and CD14dimCD16⁺ monocytes, T cells, and natural killer cells that were positive for CellROX Green by a mean 1.0-fold, 1.25-fold, 0.9-fold, 1.05-fold, 1.05-fold, respectively, compared with the sham-control session in individuals with no history of smoking or vaping (Figure 2A). Similar, nonsignificant trends were found in neutrophils, CD14²⁺CD16⁻ monocytes, and B cells of individuals with no history of smoking or vaping (Figure 2A). Compared with sham control, a single vaping session did not affect other measures of COS in cell subtypes of individuals with no history of smoking or vaping (Figure 2A and C) or individuals who currently used ECIGs (Figure 2B and D) and individuals who currently smoked TCIGs (data not shown).

Figure 2. Impact of Acute Exposure to a Single Vaping Session on Measures of Cellular Oxidative Stress (COS) in Immune Cell Subtypes by Group.

Peripheral blood mononuclear cells and flow cytometry were used to determine expression measures of COS among smoker groups at baseline and after a single exposure to to electronic cigarette vaping with an electronic cigarette with 5% nicotine (labeled as vaping) or sham vaping (puffing on a straw labeled as sham) among immune cell subtypes. Gating strategies for viability dye and antibody staining for CD45⁺ immune cells and immune cell subtypes such as CD45⁺CD15⁺CD16⁺CD14⁻hi-SSC neutrophils, CD45⁺CD14²⁺CD16⁻ classic, CD45⁺CD14²⁺CD16⁺ intermediate, CD45⁺CD14_dimCD16⁺ nonclassic monocytes, CD45⁺CD3⁺CD4⁺ T cells, CD45⁺CD3⁺CD8⁺ T cells, CD45⁺CD3⁻CD56⁺CD16⁺ natural killer (NK) cells, and CD45⁺CD19⁺ B cells have previously been described.¹ The value of each measurement (percentage of positive cells) after the intervention (sham vs vaping) was normalized by the value of each measurement at baseline within each person and was expressed as fold to the baseline value. Error bars indicate standard errors. The dotted lines indicate preexposure baseline.

^aP < .05

^bP < .01

Discussion

To our knowledge, this is the first study to report that using an ECIG during a single vaping session compared with sham control results in an increase in oxidative stress levels among several immune cell subtypes in otherwise healthy, young people with no history of smoking or vaping. Interestingly, a similar vaping session did not increase COS in individuals who currently used TCIGs or ECIGs, in whom baseline oxidative stress levels were already increased.¹

The major limitation of this study is the small sample size. Strengths of this study include the detection of COS in immune cells within the same person preintervention and postintervention. Our findings have implications for young people who have pivoted from TCIG smoking to ECIG vaping, believing, erroneously, that ECIGs are harmless.⁶ Although long-term ECIG use is associated with elevations in oxidative stress,¹ our results show that even a single vaping session can induce similar changes in individuals with no history of smoking or vaping. Intermittent increases in oxidative stress in individuals who used ECIGs experimentally may lead to significant future pathologies, including cardiovascular, pulmonary, and neurological diseases, with uncertain future adverse effects.

Supplement 1.

Trial protocol.

Supplement 2.

eMethods. Study population.

Supplement 3.

Data sharing statement.