Nicotine, Carcinogen, and Toxin Exposure in Long-Term E-Cigarette and Nicotine Replacement Therapy Users

E-cigarettes (1), which produce an aerosol by heating a solvent (e-liquid) usually containing nicotine through a battery-powered heating element, are becoming increasingly popular. Unlike smoked tobacco, e-cigarettes can deliver nicotine to the respiratory tract without combustion (2). Despite this possible advantage, health concerns for e-cigarettes remain about potential cytotoxicity; delivery of carcinogens (3), including carbonyls (4, 5), tobacco-specific N-nitrosamines (TSNAs) (6), and heavy metals (4); effects on cardiovascular and respiratory function and inflammatory effects (7); and nicotine delivery (8). Data on the long-term effects of e-cigarettes are needed to accurately assess risk and inform health professionals encountering e-cigarette users (9). Most studies to date have examined toxin concentrations in e-liquids or aerosols (4, 6) using cell-line or animal models (7). However, these models may not provide accurate information because user characteristics, together with device characteristics and their interactions, determine actual body-level exposure and thus potential health consequences (10). Three studies that have assessed such exposure found lower levels for carcinogens, including TSNAs, in recent former smokers of e-cigarettes than in a historic sample of smokers of combustible cigarettes (11); these studies also found reductions in toxins over a 2- or 4-week period in smokers switching to e-cigarettes with or without concurrent use of combustible cigarettes (12, 13). However, none of the studies involved long-term users, which is important given observed learning effects in e-cigarette use (14, 15), or included real-world control groups to reduce the risk for confounding when interpreting the results of observational studies. Users of nicotine replacement therapy (NRT) (which includes chewing gum and adhesive patches), would be an appropriate control. Dual use of combustible cigarettes and either e-cigarettes or NRT is common, and long-term use of both types of products has been reported (16, 17). They have been advocated to reduce the harms and risks associated with combustible tobacco (18). However, unlike e-cigarettes, the NRT safety profile is well-established (19) and NRT effectiveness for smoking cessation through initial partial (20) or complete substitution (21) has been shown. Therefore, NRT is recommended as a harm reduction strategy in several countries (22). Although longitudinal cohort studies and randomized, controlled trials will provide the best data to answer questions about the safety and efficacy of e-cigarettes for smoking cessation, these designs are time- and resource-intensive. In the absence of long-term data, a more pragmatic approach is to compare smokers and former smokers with or without concurrent e-cigarette use in real-life settings. This study aimed to address the gap in the literature by measuring biomarker levels in long-term e-cigarette users compared with an appropriate controlNRT users. Specifically, this study assessed whether long-term e-cigaretteonly, NRT-only, dual combustible cigarettee-cigarette, or dual combustible cigaretteNRT use is associated with differences in metabolites of nicotine, TSNAs, and volatile organic compounds (VOCs) compared with combustible cigaretteonly use. Methods Study Design and Procedure This cross-sectional study was done in London, United Kingdom, from January 2014 to June 2014. It evaluated the range of toxin levels measured in smokers and former smokers with or without concurrent long-term use of e-cigarettes or NRT. The study methodology has been described elsewhere (23). Briefly, participants visited the laboratory for a single session, lasting 30 minutes, after abstaining from eating, drinking, or using combustible cigarettes or other nicotine products for an hour before their visit to standardize assessment. At the laboratory, after providing written consent, participants completed a short questionnaire assessing sociodemographic, smoking, and product use characteristics and provided breath, saliva, and urine samples. Exhaled air was assessed for carbon monoxide with a breathalyzer (Micro IV Smokerlyzer, Bedfont Scientific). In addition, 2 saliva samples were collected with sterile dental rolls (Salivette, Sarstedt) that participants were asked to gently chew for about 2 minutes or until saturated. Urine was collected in a sealable, sterilized cup by participants on site and transferred by staff into cryovials. Urine and saliva samples were then kept frozen at 20C until they were shipped in dry ice to laboratories at Roswell Park Cancer Institute (Buffalo, New York) and the Centers for Disease Control and Prevention (Atlanta, Georgia) for analysis. All participants were reimbursed for time and travel (25). The study was approved by the University College London Ethics Committee (project ID 0483/002). Participants Participants were purposively recruited in the greater London area using various methods to increase sample diversity, including newspapers and online advertisements, posters in pharmacies, and the use of marketing companies. They had to be ever smokers and to meet the following eligibility criteria: Current smokers had to smoke an average of 5 or more combustible cigarettes per day for at least 6 months, and former smokers had to have stopped using tobacco products (including combustible cigarettes, water pipes, cigars, and such smokeless products as snus or chewing tobacco) for at least 6 months. Because we sought to evaluate the effect of long-term use of noncombustible nicotine delivery devices (NRT and e-cigarettes), smokers (that is, dual combustible cigarettee-cigarette or combustible cigaretteNRT users) and former smokers (that is, e-cigaretteonly or NRT-only users) had to have been using these products at least weekly for 6 months or more (users of nicotine-free products, such as e-liquid without nicotine, were excluded). In practice, however, participants used products daily as indicated by latency to last product use across groups (combustible cigarettesonly users, 1.4 hours; combustible dual cigaretteNRT users, 4.3 hours; combustible dual cigarettee-cigarette users, 1.3 hours; NRT-only users, 24 hours; and e-cigaretteonly users, 5.4 hours). Product use was verified by asking participants to bring in the NRT or e-cigarette that they were currently using, and smoking status was verified with carbon monoxide readings (10-ppm cutoff) (24). We excluded persons who used both NRT and e-cigarettes as well as those who were younger than 18 years; were pregnant; had a history of heart or lung disease; or had bleeding gums, illness, or an active infection within 24 hours of their scheduled appointment. Measures Biomarkers of Exposure Level of nicotine exposure was measured to assess effectiveness of nicotine delivery products by using 2 methods. Saliva samples were analyzed for nicotine, and its major metabolite, cotinine, using an established gas chromatography method (25, 26). Urine samples were analyzed for main nicotine metabolites to derive total nicotine equivalents and for minor tobacco alkaloids using validated tandem mass spectrometry (27, 28). Levels of urinary TSNA and VOC metabolites were measured using either liquid chromatography/atmospheric pressure ionization/tandem mass spectrometry (29) or ultra-high performance liquid chromatography coupled with electrospray ionization and tandem mass spectrometry (30) to assess the potential risks of nicotine delivery products. Although we assessed a comprehensive battery of metabolites (Appendix Table 1), we focus here on well-established metabolites of compounds that are known to contribute significantly to smoking-related toxicologic and carcinogenic risks (3139) (Table 1). All urinary and salivary biomarkers were analyzed by the Centers for Disease Control and Prevention and Roswell Park Cancer Institute, respectively. Appendix Table 1. Urinary and Saliva Biomarker Levels, by Group* Table 1. Major Toxicants and Carcinogens Related to Tobacco Use Covariates Sociodemographic characteristics (age, sex, ethnicity, education, and marital status) were assessed in addition to self-reported recently resolved physical illness (chest infection, cold or flu, sore throat, or fever) and subjective well-being (happiness and satisfaction, both assessed with established single-item measures) (40). Salivary C-reactive protein level was used as a marker of inflammation (and thus potential health problems) and analyzed with an enzyme-linked immunosorbent assay (Salimetrics Europe) (41). Smoking characteristics, including current and past daily combustible cigarette consumption as a measure of dependence for smokers and former smokers, respectively; age at which participants had started smoking; and the proportion of family members or friends who smoke were assessed to gauge environmental tobacco smoke exposure. Statistical Analysis Because this was a cross-sectional study, exposure biomarkers, including metabolites of known tobacco-related carcinogens and toxins, were used as proxies for future disease risk. Previous research on the association of the carcinogenic metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) with lung cancer suggests that medium to large reductions in NNAL levels (Cohen f= 0.25 to 0.40) would result in an appreciable reduction in risk (42) and could thus be considered clinically meaningful in magnitude and warrant further investigation (43). A priori power calculation showed that 180 participants (36 per group) would provide 90% power to detect between-group differences of a medium effect size (Cohen f= 0.3) in NNAL levels when comparing 5 groups by using analysis of variance (44). However, this calculation did not account for multiple outcomes being tested, and based on 35 biomarker outcomes reported here, power to detect such an effect size across all biomarkers would have been reduced to 54%. The sample size therefore onl