11

Respiratory Diseases

Smoking of combustible tobacco products is the number one cause of chronic obstructive pulmonary disease (COPD) worldwide. Although the proportion of smokers has decreased over the past 25 years, approximately 1.1 billion people continue to smoke as of 2015 (Rabe and Watz, 2017). COPD leads to more than 3 million deaths per year worldwide, with only ischemic heart disease and cerebrovascular disease causing more deaths. Individuals who smoke also have an increased risk of sleep apnea and asthma exacerbations (Jayes et al., 2016). Respiratory complications from smoking can be further confounded by the increase in cardiovascular disease in individuals who smoke (Rabe and Watz, 2017).

In addition to the adverse respiratory health effects caused by smoking combustible tobacco products, secondhand smoke exposure has been reported to be associated with significant respiratory morbidities in non-users (Jayes et al., 2016). Tobacco smoke exposure has been shown to increase the severity of asthma exacerbations in children exposed to secondhand smoke (Merianos et al., 2016). Exposure to tobacco smoke in utero has been associated with abnormalities in lung development and small airway dysfunction in school-age children, manifested by reductions in forced expiratory volume in 1 second (FEV1) and forced expiratory flow 25–75 percent (FEF25–75 percent) (den Dekker et al., 2015; Duijts et al., 2012; Hayatbakhsh et al., 2009). A study in China found that school-age children exposed to secondhand smoke had increased cough and decreased lung function compared with children not exposed to secondhand smoke (He et al., 2011), and a study from Finland found that

children of mothers who smoked combustible tobacco cigarettes during pregnancy were more likely to have increased airway resistance than children of mothers who did not smoke (Kalliola et al., 2013). Postnatal exposure to tobacco smoke also has been associated with an increased risk of wheeze and upper and lower respiratory tract illnesses in exposed children compared with unexposed children (Jayes et al., 2016).

Currently there is a lack of information regarding the short- and long-term effects of e-cigarettes on the respiratory system. This is due in part to the relative newness of the delivery system, the vast assortment of devices being used, and the variety of nicotine concentrations and flavorings that are currently available. Nevertheless, exposure of the lungs to various components of the e-cigarette aerosol could potentially damage the respiratory system or worsen preexisting lung disease through a variety of mechanisms (see Figure 11-1). For example,

nicotine-containing e-cigarette aerosols have the potential to adversely impact several host defense mechanisms in the lungs. In a murine model, α7 nicotinic acetylcholine receptors (α7 nAChRs) were shown to regulate cystic fibrosis transmembrane conductance regulator (CFTR) activity in the airways. Exposure to nicotine downregulated α7 nAChR activity, which in turn impaired CFTR function, causing impaired mucociliary clearance (MCC) (Maouche et al., 2013). In humans, CFTR dysfunction has been shown to be associated with the development of COPD and asthma hyperresponsiveness (Saint-Criq and Gray, 2017). Exposure to nicotine in tobacco smoke and e-cigarette aerosols also has been reported to impair cough (Dicpinigaitis, 2017; Dicpinigaitis et al., 2006; Sitkauskiene and Dicpinigaitis, 2010). Furthermore, nicotine has been shown to downregulate Th1 immune responses to lipopolysaccharide (Yanagita et al., 2012), consistent with an immunomodulatory effect of nicotine on viral and bacterial clearance.

Independent of nicotine, exposure to particulates and flavorings in e-cigarette aerosols could also potentially impair lung function. The presence of ultrafine particles has been measured in the aerosols of e-cigarettes (Laube et al., 2017), and particulates in the submicron range have the potential to damage airways and lung parenchyma. As noted in Chapter 3, the health risks from exposure to particles will depend on their nature, not simply their size. Nevertheless, certain ultrafine particles, which encompass particle sizes less than 100 nm, can cause DNA damage, induce pro-inflammatory cytokine expression, and adversely affect the immune system through the production of free oxygen radicals (Li et al., 2016). In addition, inhalation of ultrafine particles has been reported to increase the rate of asthma exacerbations (Li et al., 2016). Flavorings in e-cigarettes may also alter cellular redox balances in the airways by increasing pro-inflammatory cytokines (Lerner et al., 2015), and high temperatures generated by e-cigarette devices may cause formation of formaldehyde, leading to toxic effects on the lungs (Geiss et al., 2015).

In established smokers who are trying to quit or reduce combustible tobacco use, e-cigarettes may be less deleterious to the respiratory system when compared with exposure to combustible tobacco smoke (see Chapter 18). However, initiation of e-cigarette use by a person who has never smoked may cause harm to the respiratory system compared with never using e-cigarettes, particularly if initiation of e-cigarettes occurs at a young age. Therefore, understanding the health effects of e-cigarettes is dependent on the context of age, current and prior use of combustible tobacco products, and whether the user has preexisting lung conditions such as asthma and COPD. In addition, there is a need to examine the short- and long-term effects of secondhand and thirdhand e-cigarette aerosols on the respiratory health of non-users, who may inhale or come

in contact with exhaled mainstream aerosol, which can settle on hard surfaces. Infants and preschool children who live with e-cigarette users may be at higher risk for secondary exposures because this age group spends much of their time in the residence of the e-cigarette user. Finally, exposure of the dual user to both combustible tobacco products and e-cigarette aerosols may cause unique health risks to the respiratory system.

CHARACTERIZATION OF DISEASE ENDPOINTS AND INTERMEDIATE OUTCOMES

In studying the effects of e-cigarette use on respiratory disease endpoints, an important question is whether or not e-cigarette use by itself can lead to the development of chronic respiratory conditions such as asthma and COPD or if e-cigarette use can worsen preexisting lung conditions compared with people who do not smoke. Additionally, researchers should determine if substitution of e-cigarettes for combustible tobacco use lessens the development of chronic respiratory conditions or lessens progression of preexisting lung conditions compared with people who continue to smoke. Because these respiratory disease endpoints may take years or even decades to realize, it becomes necessary to measure intermediate outcomes that may predict a disease state. The intermediate outcomes most relevant to the clinician include measurements of lung function and lung structure. The most common measurements of lung function include forced vital capacity (FVC), FEV1, FEV1/FVC ratio, and FEF25–75 percent, with the latter three the most useful in detecting presence and progression of obstructive lung diseases, such as asthma and COPD. These measurements are easily obtainable using spirometry. Body plethysmography can be used to detect an increase in residual volume, which can correlate with worsening airflow obstruction. In addition, impulse oscillometry can be used to detect changes in large and small airway resistance, and may be more sensitive than spirometry in detecting reversibility of airway obstruction in people with COPD (Saadeh et al., 2015). Structural changes in the lung such as the development of emphysematous changes or mucus plugging can be determined using computed tomography (CT) of the chest. Ultra-low-dose CT (Messerli et al., 2017), and more recently, MRI of the chest, has been shown to be an alternative modality to conventional chest CT in assessing COPD changes (Saadeh et al., 2015; Washko et al., 2012). Finally, standardized respiratory questionnaires can be helpful in evaluating outcomes; however, instrument responsiveness may differ among questionnaires (Puhan et al., 2006). Research on other intermediate outcomes in respiratory health should include the effect of e-cigarette aerosols, with and without nicotine, on cough reflex sensitivity, urge to cough, and nasal MCC because

cough and MCC are integral defense mechanisms that help clear pathogens and environmental pollutants from the lungs and sinuses (Chatwin et al., 2003; Lee et al., 2017; Tarrant et al., 2017).

Quantification of inflammatory cell numbers from bronchoalveolar lavage (BAL) (Levanen et al., 2016; Siew et al., 2017) and measurement of pro-inflammatory cytokines from bronchial biopsies (Shields et al., 2017) could be used as intermediate respiratory endpoints to assess inflammation in the lower respiratory tract inflammation caused by e-cigarette use. In addition, combustible tobacco smoke has been shown to alter microbiome diversity; therefore, examination of sputum, nasal, and pharyngeal microbiome diversity may also help predict the impact of e-cigarette use on respiratory health (Diao et al., 2017). Other intermediate outcomes that could be used as markers of respiratory health include self-reported wheeze, bronchitis, shortness of breath, mucus production, other respiratory symptoms, and quality of life measurements.

OPTIMAL STUDY DESIGN

Since the potential health effects of e-cigarettes on the respiratory system are not completely understood, randomized controlled trials (RCTs) would not be appropriate at this time. Alternatively, prospective cohort studies that assess respiratory health outcomes in e-cigarettes users compared with combustible tobacco users and dual users could help determine the risks and benefits of using e-cigarettes. In addition, RCTs testing the efficacy of e-cigarette substitution as a method of smoking cessation in smokers unable to quit using nicotine replacement therapy (NRT) could concurrently measure lung function, lung structure, lung symptoms, and quality of life in individuals substituting e-cigarettes for combustible tobacco products. These additional studies could provide valuable information regarding the respiratory health effects of e-cigarette substitution on established smokers and help determine if switching completely or partly to e-cigarettes from combustible tobacco products in people with preexisting lung disease can alter progression or stability of lung disease.

Prospective cohort studies in adolescents and young adult e-cigarette users without a history of combustible tobacco product use should be performed to determine the likelihood of e-cigarette use leading to the development of chronic respiratory symptoms or decline in lung function. Furthermore, since asthma is a common respiratory disease of childhood, it is also important to determine if adolescents and young adults with asthma are at increased risk for asthma exacerbations and a more rapid decline in lung function when using e-cigarettes. Potential confounding factors, such as dual tobacco or cannabinoid use, exposure to secondhand smoke, and prior history of tobacco use, could introduce bias into the

comparisons across exposure groups and need to be considered. Rigorous, objective assessment of the spectrum of endpoints, including lung function, respiratory symptoms, and cardiovascular and other comorbidities would also be essential to these studies.

QUESTIONS ADDRESSED BY THE LITERATURE

Due to the relatively recent widespread acceptance of e-cigarettes, there is a lack of understanding regarding the positive and negative effects of e-cigarettes on respiratory health. This is due in part to the paucity of long-term observational studies of adolescent/young adult never smokers who initiate e-cigarette use and observational studies and RCTs of adult smokers who switch to e-cigarettes for smoking cessation. Human studies are also needed that examine how exhaled mainstream aerosols affect the respiratory system of non-users when inhaled. As previously noted, exposure to these aerosols may disproportionately impact infants and children in the homes of indoor e-cigarette users because the very young often spend the majority of their time in this environment. However, since e-cigarettes, unlike combustible tobacco products, lack substantial sidestream emissions, it is unclear how detrimental exposure to secondhand e-cigarette emissions is to the non-user.

Further investigations into the effects of e-cigarette aerosols on the lung defense mechanisms such as cough, MCC, and the innate and adaptive immune system are needed. In addition, a better understanding of the impact of particle size on the development of DNA damage in respiratory cells is needed as is the relationship between flavorings and development of reactive oxygen species.

CLINICAL AND EPIDEMIOLOGICAL STUDIES IN HUMANS

Effects on Users of Combustible Tobacco Products

The literature search identified 17 studies that examined respiratory or pulmonary outcomes in people using e-cigarettes (see Table 11-1). Subjects in these studies include adult users of combustible products who switch to e-cigarettes completely or become dual users and include subjects with or without preexisting respiratory disease. Outcomes in the studies include standard measures of function ranging from self-reported symptoms of cough to asthma to exhaled carbon monoxide or nitric oxide. Six of these studies were from the same study group (Campagna et al., 2016; Cibella et al., 2016; Polosa et al., 2014a,b, 2016a,b). Three of these studies were observational studies in which the subject population included smokers not intending to quit. These subjects were invited to

switch to first-generation e-cigarettes (Campagna et al., 2016; Cibella et al., 2016; Polosa et al., 2014b). Cibella and colleagues (2016) reported significant improvement in self-reported respiratory symptoms of cough/phlegm at 52 weeks in smokers who switched completely to e-cigarettes (18 of 130 subjects) and a significant increase in FEF25–75 percent, but not in FEV1 or FVC. No difference in lung function was found in dual users at 52 weeks (Cibella et al., 2016). In a similar study population of smokers not intending to quit, Campagna and colleagues (2016) found significant decreases in the fractional concentration of carbon monoxide in exhaled breath (FeCO) in smokers who switched completely to e-cigarettes (18 of 134 subjects) and significant increases in the fractional concentration of nitric oxide in exhaled breath (FeNO) at 52 weeks. Polosa and colleagues (2014b) reported on 40 smokers not intending to quit, 17 of whom were lost to follow-up, and found that when invited to use e-cigarettes, 5 of the 40 switched completely to e-cigarettes at 24 months.

In two studies from Polosa and colleagues (2014a, 2016a), they identified retrospectively 18 mild to moderate asthmatic smokers who switched to e-cigarettes (either single or dual users). They reported an improvement in FEV1, performance in the methacholine challenge test, and asthma control questionnaire but no change in asthma exacerbations when these subjects were followed prospectively over a 12-month period (Polosa et al., 2014a, 2016a). In a similar study design, Polosa and colleagues (2016b) identified patients with COPD from medical records who switched to e-cigarettes (single or dual users) and reported that they had significantly fewer COPD exacerbations. D’Ruiz and colleagues (2017) reported on pulmonary function tests in smokers who were switched to e-cigarettes for 5 days and found no significant difference in lung function between the groups.

These studies suggest that smokers with preexisting lung conditions such as asthma and COPD may experience some benefits from switching to e-cigarettes. As reported in the Polosa and colleagues (2016a,b) studies, such benefits may include an increase in FEV1, improved performance in a methacholine challenge test and in asthma control, and a decrease in COPD exacerbations. However, a limitation of these studies is that they were performed in a small number of subjects selected retrospectively. In addition, a reasonably high-quality RCT was negative: Cravo and colleagues (2016), in a clinical study that recruited subjects from two centers in the United Kingdom, reported no difference in lung function in subjects who switched to e-cigarettes. Their study had two cohorts. In both cohorts, smokers were randomized to either change to e-cigarettes containing 2 percent nicotine (with or without menthol flavoring) or to continue smoking. The authors reported no significant changes in pulmonary function tests after 12 weeks between the two groups. In this study,

TABLE 11-1 Clinical and Epidemiological Studies in Humans

NOTES: 6-MWD = 6-minute walk distance; ACQ = asthma control questionnaire; AE = adverse event; AHR = airway hyperresponsiveness; ANCOVA = analysis of covariance; ANOVA = analysis of variance; AOR = adjusted odds ratios; BHR = bronchial hyperresponsiveness; BL = baseline; C₅ = cough reflex sensitivity; CAT = COPD Assessment Test; CO = carbon monoxide; COPD = chronic obstructive pulmonary disease; Cu = urge to cough; eCO = exhaled carbon monoxide; ECG = echocardiogram; FeCO = fractional concentration

smokers with respiratory conditions were excluded from the study and subjects in the e-cigarette randomized arm, although encouraged not to use combustible tobacco cigarettes, did often report dual use (Cravo et al., 2016).

Taken together the majority of studies in the literature examining respiratory outcomes of e-cigarette use and their benefit on respiratory function in current smokers come from the same region of Italy, which limits generalizability of their results. In addition, with the exception of the study by Cravo and colleagues (2016), the sample sizes were generally small and subjects were selected retrospectively.

Acute Exposures

Two studies focused on the short-term effects of e-cigarettes on exhaled breath measurements (FeCO and FeNO) and pulmonary function tests. The first study examined the effects of nicotine-free e-cigarettes on lung function and exhaled breath measurements. Ferrari and col-

of carbon monoxide; FeNO = fractional concentration of nitric oxide; FEF25–75% = forced expiratory flow at 25–75 percent of the pulmonary volume; FEV1 = forced expiratory volume; FVC = forced vital capacity; GOLD Stages 1–4 = Global Initiative for Chronic Obstructive Lung Disease Stages of COPD (1 = mild, 2 = moderate; 3 = severe; 4 = very severe); MCC = mucociliary clearance; NF = nicotine free; NO = nitric oxide; PEF = peak expiratory flow; SHS = secondhand smoke; SNOT-22 = Sino-Nasal Outcome Test; SoB = shortness of breath.

leagues (2015) recruited 10 smokers and 10 non-smokers and found no significant decline in lung function after 5 minutes in the subjects using nicotine-free e-cigarettes by contrast to subjects who smoked combustible tobacco cigarettes. Vardavas and colleagues (2012) recruited healthy smokers and found that after 5 minutes of using a nicotine-containing e-cigarette, airway flow resistance increased and FeNO decreased from baseline. Although the mechanisms underlying the lower FeNO in e-cigarette users are unclear, smokers also have been shown to have low FeNO levels compared with non-smokers (Malinovschi et al., 2012; Torén et al., 2006). This suggests that the mechanisms that cause lower FeNO in e-cigarette users are similar to those that cause lower FeNO levels in smokers. Although higher FeNO levels have been demonstrated in people with eosinophilic-induced asthma and are considered a marker of airway inflammation (Malinovschi et al., 2012), studies of subjects with other respiratory conditions including cystic fibrosis (CF) have reported lower FeNO levels, possibly associated with impaired CFTR function (Korten et al., 2018).

Cough and Mucociliary Clearance

Airway exposure to nicotine has also been implemented as a causal factor in inhibiting cough and MCC defenses (Dicpinigaitis et al., 2016a; Laube et al., 2017; Maouche et al., 2013). Specifically, nicotine may modulate perceptual and motor responses to irritant cough stimulants (capsaicin), inhibiting the urge to cough (Davenport et al., 2009). Four studies reported on the effects of e-cigarettes on cough and nasal MCC (Dicpinigaitis, 2017; Dicpinigaitis et al., 2016a,b; Kumral et al., 2016). In a randomized single-blind clinical trial, Kumral and colleagues (2016) used Sino-Nasal Outcome Test (SNOT-22) scores and measured nasal MCC of subjects recruited from a smoking cessation clinic in which they were assigned to either e-cigarettes or non-e-cigarette cessation therapy. At 3 months, subjects assigned to the e-cigarette group had significantly worse sino-nasal symptoms and nasal MCC than subjects assigned to the non–e-cigarette group (Kumral et al., 2016). Two studies from Dicpinigaitis and colleagues (2016a,b) recruited healthy adult nonsmokers. Subjects were challenged with capsaicin at baseline and then at 15 minutes and 24 hours after a short exposure to nicotine-containing or non–nicotine-containing e-cigarettes. They found that urge to cough as measured by capsaicin challenge was depressed at 15 minutes following nicotine-containing e-cigarettes, but not nicotine-free e-cigarettes. At 24 hours after nicotine-containing e-cigarette use, cough reflex sensitivity returned to baseline (Dicpinigaitis et al., 2016b). Dicpinigaitis and colleagues (2016a) also reported that nicotine-containing e-cigarettes caused a decrease in cough reflex sensitivity (C₅), analyzed using mixed-effects modeling, at 15 minutes after nicotine-containing e-cigarette use but not after nicotine-free e-cigarette use. Dicpinigaitis (2017) again highlighted the role of nicotine causing centrally mediated suppression of cough in a study in which he reported suppression of cough at 15 minutes after capsaicin challenge in both combustible tobacco cigarette users and users of nicotine-containing e-cigarettes. Following cessation of combustible tobacco cigarette smoking, this centrally mediated cough reflex returned (Dicpinigaitis, 2017).

Respiratory Symptoms in Adolescents

Four studies examined respiratory symptoms in adolescents using or who have used e-cigarettes. Using self-reported questionnaires from participants in the Southern California Children’s Health Study, McConnell and colleagues (2017) found a significant association between increased rates of chronic bronchitis symptoms among past, but not current, e-cigarette users over the previous 12 months. Cho and Paik (2016), using a Web-based questionnaire in a population of high school students from

South Korea, found that students who used e-cigarettes were more likely to have a self-reported clinical diagnosis of asthma and were more likely to have been absent from school due to severe asthma symptoms. Using an anonymous questionnaire with Chinese adolescents in Hong Kong, Wang and colleagues (2016) reported a higher rate of respiratory symptoms in those who used e-cigarettes regardless of previous or current history of smoking and observed that adolescents who used e-cigarettes had more days absent from school because of asthma. Choi and Bernat (2016) examined the prevalence of ever and past 30-day use of e-cigarettes by adolescents, using the 2012 Florida Youth Tobacco Survey. They reported an association between past 30-day e-cigarette use and having an asthma exacerbation in adolescents with asthma. Interestingly, adolescents with asthma in this study were more likely to have used e-cigarettes ever and in the past 30 days compared with adolescents not diagnosed with asthma.

IN VIVO ANIMAL STUDIES AND IN VITRO MECHANISTIC STUDIES

Animal studies in combination with in vitro studies have provided some unique insights into the potential health effects associated with e-cigarette use. Larcombe and colleagues (2017) exposed 4-week-old female BALB/c mice to 8 weeks of either tobacco smoke or propylene glycol (PG) or glycerol e-cigarette solutions with and without nicotine. They found that mice exposed to tobacco smoke had increased pulmonary inflammation and changes in pulmonary function, including methacholine hyperresponsiveness. Although inflammation was not increased in the e-cigarette–exposed mice, pulmonary function abnormalities were found. A limitation to the study is that they excluded male mice from analysis.

Garcia-Arcos and colleagues (2016) examined the effects of aerosolized nicotine-free and nicotine-containing e-cigarette fluid via inhalation in mice and normal human airway epithelial cells. Exposure in mice was for 1 hour per day for 4 months. Human bronchial epithelial (HBE) cells were cultured at an air–liquid interface with exposure to e-cigarette aerosols or nicotine solutions. Exposure to inhaled nicotine-containing e-cigarette fluids triggered effects normally associated with the development of COPD, including increased airway hyperreactivity, distal airspace enlargement, mucin production, and cytokine and protease expression. Exposure to nicotine-free e-cigarettes did not affect these lung parameters, suggesting effects were nicotine dependent in the mouse lung. These effects were also nicotine dependent in human airway cells in culture, further suggesting that inhaled nicotine contributes to airway and lung

disease in addition to its addictive properties. Exposure of HBE cells to nicotine-containing e-cigarette fluids also demonstrated impaired ciliary beat frequency, airway surface liquid volume, cystic fibrosis transmembrane regulator, and ATP-stimulated K⁺ ion conductance and decreased expression of FOXJ1 and KCNMA1. The major concerns for this study include the matter of aerosolization and the dose delivered to animals by inhalation compared with human use, as well as the dose delivered to cells in culture versus actual exposure conditions in vivo (Garcia-Arcos et al., 2016).

Acute exposure to e-cigarettes compared with combustible tobacco cigarette smoke has been studied by Husari and colleagues (2016). Mice were exposed for 6 hours per day to air, e-cigarette, or combustible tobacco cigarette smoke for 3 days with higher particulate levels for e-cigarettes compared with combustible tobacco cigarette smoke. Human alveolar cells (A549) in culture were also exposed to various concentrations of e-cigarette aerosol and combustible tobacco cigarette smoke extracts. The authors found a significant increase in interleukin-1β (IL-1β) with exposure to e-cigarette, while combustible tobacco cigarette smoke resulted in significant increases in IL-1β, IL-6, TNF-α expression, and oxidative stress. TUNEL staining demonstrated significant cell death with combustible tobacco cigarette smoke, but not with exposure to e-cigarettes. Concerns about this study include the manner of exposure delivery to animals and the relevance of the A549 cell test results to the assessment of human implications for health (Husari et al., 2016).

Lim and Kim (2014) examined e-cigarette cartridge solution and its potential to aggravate allergen-induced airway inflammation and hyperresponsiveness in BALB/c mice. These investigators used diluted e-cigarette cartridge solution, which was delivered to mice by intratracheal instillation two times a week for 10 weeks. The mice had been previously sensitized to ovalbumin (OVA) by intratracheal, intraperitoneal, and aerosol allergen challenge. E-cigarette exposure increased infiltration of inflammatory cells, including eosinophils, into airways; enhanced the asthmatic AI and airway hyperresponsiveness; and stimulated cytokine production of IL-4, IL-5, and IL-13, as well as OVA-specific IgE production. These data suggest e-cigarette solutions can exacerbate allergy-induced asthma symptoms. This study is limited by its use of intratracheal instillation of dilute e-cigarette solution rather than true delivery of e-cigarette exposure by inhalation.

Hwang and colleagues (2016) examined the effects of e-cigarette inhalation on immune function. Mouse inhalation of e-cigarette aerosols was done 1 hour daily for 4 weeks, leading to alterations in inflammatory markers within the airways and elevation of an acute-phase reactant in serum. Exposure of human epithelial cells at the air–liquid interface

to aerosols from an e-cigarette device resulted in dose-dependent cell death; in mice, reduced antimicrobial activity against Staphylococcus aureus in epithelial cells, alveolar macrophages, and neutrophils were observed. The authors concluded that inhalation of e-cigarette aerosols alters immunomodulatory cytokines in the airways of mice and increases markers of inflammation in BAL and serum, thus enhancing the virulence of Staphylococcus aureus. Although observations of e-cigarette impact are similar in mice and cells in culture, the actual mechanisms based on dose are difficult to ascertain (Hwang et al., 2016).

Additional studies, by Sussan and colleagues (2015), also questioned how e-cigarettes may impair antibacterial and antiviral defenses in mice. They found e-cigarette aerosol exposure for 2 weeks produced a significant increase in oxidative stress and moderate macrophage-mediated inflammation, and significantly impaired pulmonary bacterial clearance, compared with air-exposed mice, following an intranasal infection with Streptococcus pneumonia. For mice infected with influenza A virus, e-cigarette exposure was associated with increased lung viral titers and enhanced virus-induced illness and mortality. These findings demonstrate that e-cigarettes may impair the immune response and enhance susceptibility to bacterial and viral infections (Sussan et al., 2015).

Laube and colleagues (2017) exposed 10-week-old male mice to e-cigarette aerosol containing PG alone or PG in combination with nicotine for 20 minutes per day for either 1 or 3 weeks. Following exposure, mice were examined for MCC using technectium-labeled sulfur colloid with clearance of the colloid determined using an X-SPECT gamma camera. The research showed that daily exposure for 3 weeks to PG and nicotine slowed MCC compared with exposure to PG alone. This finding supports the potential biological plausibility of the previous study by Sussan and colleagues (2015), which also used mice and showed impaired bacterial clearance in the lungs of mice. Together, these studies provide evidence that exposure to e-cigarette aerosols during adolescence and early adulthood is not harmless to the lungs and can result in significant impairments in lung function even in the absence of lung inflammation.

Toxicity, oxidative stress, and inflammatory response in mice and human airway epithelial cells were examined by Lerner and colleagues (2015). E-cigarette exposure in C57BL/6J mice increased pro-inflammatory cytokines, while diminishing glutathione levels in the lungs, critical in maintaining a balance of cellular redox in the lungs. E-cigarette aerosol exposure of human airway epithelial cells (H292) in an air–liquid interface system resulted in increased secretion of inflammatory cytokines IL-6 and IL-8. Delivery of unaerosolized e-liquids was also found to be oxidative dependent on flavor additives. They found sweet or fruit flavors to be stronger oxidizers than tobacco flavors. Thus, exposure to

e-cigarette aerosols/e-liquids produces measurable oxidative and inflammatory responses in lung cells and tissues that might lead to unrealized health consequences. Concerns about this study are minimal, but include the methods of delivery to cells in culture and the extrapolation of in vitro results to humans.

E-cigarette exposure has been found to have potential implications on the larynx as well. Salturk and colleagues (2015) found that exposure of Wistar albino rats to e-cigarette aerosol for 1 hour per day for 4 weeks caused hyperplasia and metaplasia of the laryngeal mucosa of rats, but this finding was not statistically significant. This study, although interesting, is inconclusive as to the relevance of how possible health effects to the larynx should be considered in e-cigarette use. Laube and colleagues (2017) examined MCC changes in C57BL/6 mice after 3 weeks of daily exposure and found that young adult male mice exposed to PG alone had significantly higher MCC than mice exposed to nicotine/PG aerosol. This study suggested that chronic exposure to nicotine-containing e-cigarette aerosols can impair airway MCC.

A 90-day inhalation study in rats, followed by a 42-day recovery period, was conducted by Werley and colleagues (2016). Exposure was done with low-, mid-, and high-dose levels of aerosols composed of vehicle (glycerol and PG mixture); vehicle and 2.0 percent nicotine; or vehicle, 2.0 percent nicotine, and flavor mixture. Daily targeted aerosol total particulate matter (TPM) doses of 3.2, 9.6, and 32.0 mg/kg/day were achieved by exposure to 1 mg/L aerosol for 16, 48, and 160 minutes, respectively. Treatment-related effects following 90 days of exposure included changes in body weight, food consumption, and respiratory rate. Also observed were dose-related decreases in thymus and spleen weights, and increased BALF lactate dehydrogenase, total protein, alveolar macrophages, neutrophils, and lung weights. This study in rats provides some insight for establishing a threshold level based on body-weight decreases at the mid-dose level for each formulation, equivalent to a daily TPM exposure dose of approximately 9.6 mg/kg/day. Histopathology changes appear to be isolated to the nasal mucosa. Concerns for this study include how to extrapolate these findings to human exposure and the relevance of the e-cigarette device used and non-respiratory parameters used for comparison. Further, lung weights and body weights are crude measures of effect.

One study reported that neonatal exposure to aerosol from nicotine-containing e-cigarettes was associated with diminished alveolar cell proliferation and impairment in postnatal lung growth (McGrath-Morrow et al., 2015).

SYNTHESIS AND CONCLUSIONS

The human observational studies examining the effect of switching to e-cigarettes (single or dual use) provide support for a finding of beneficial health effects relative to continued use of combustible tobacco products, with most favoring that conclusion. These studies were judged to be of fair quality. A major limitation of them, however, is that they are primarily from a single study group. In addition, the one RCT was negative, finding no improvement in lung function after 12 weeks in subjects who switched to e-cigarettes compared with people who continued to smoke combustible tobacco cigarettes (Cravo et al., 2016). Therefore, the committee concludes that there is limited evidence supporting improvements in lung function in smokers who switch to e-cigarettes.

Studies examining the long-term effects of e-cigarettes on the development of chronic respiratory symptoms are completely lacking due to the newness of the product. It is of importance to know whether chronic e-cigarette use by itself can cause COPD and if substitution of e-cigarettes for combustible tobacco products can prevent or slow the development of COPD in smokers who quit or reduced use of combustible tobacco products. At this time, there is a lack of well-designed epidemiological studies examining either question.

Studies examining the short-term effects of e-cigarettes indicate that nicotine-containing e-cigarettes, but not nicotine-free e-cigarettes, can have short-term adverse effects on lung defense mechanisms, including MCC, urge to cough, and cough sensitivity. These studies are of fair quality. They include subjects with and without a history of smoking and there are few-or-no credible opposing findings. These studies provide moderate evidence supporting short-term adverse effects of nicotine-containing e-cigarettes on lung defense mechanisms.

The committee identified four studies examining the effects of e-cigarette use on adolescent respiratory health—all are cross-sectional and use self-reported questionnaires. They include large groups of adolescents from three countries and reach similar results, thus providing moderate evidence of an association between respiratory symptoms in adolescents and e-cigarette use.

In non-users who are exposed to secondhand smoke and in healthy adolescents and young adult users, common respiratory endpoints can include an increase in asthma symptoms and severity and a higher prevalence of upper and greater lower respiratory tract symptoms and infections (Liu et al., 2016; Shargorodsky, 2016; Shargorodsky et al., 2015; Wilson et al., 2013). Currently, there is a lack of rigorously designed epidemiological studies examining the relationship between chronic e-cigarette use in adolescents and young adults and increased prevalence of respiratory symptoms and respiratory illnesses. There are also no epidemio-

logical studies reporting on the respiratory effects of exposure to exhaled mainstream smoke from an e-cigarette user on a non-user.

The animal studies that have examined the effects of e-cigarettes on respiratory outcomes have used different e-cigarette devices, pumps, solutions, and exposures, limiting the ability to compare results among studies. Confounding factors such as aerosol temperature and particle size have not been taken into account. These methodological differences among studies can result in differences in particle deposition in the lungs and differences in systemic absorption of particles, nicotine, and toxins, resulting in different respiratory outcomes. In addition, not all studies evaluating the effects of nicotine aerosols on lung inflammation, MCC, and lung immune responses have included biomarkers of systemic nicotine absorption, which would help to standardize exposures in animal studies. The utility of studies using whole-body exposures in animal models when examining health effects of e-cigarette aerosols is limited because this type of exposure may overestimate or underestimate an exposure in the human condition. Furthermore, in vitro cell studies would be more informative and representative of the human condition if aerosols rather than liquid e-cigarette solutions are used and if primary, instead of immortalized, cell lines are used. Despite these limitations, the animal and in vitro studies described provide additional evidence of adverse effects of e-cigarette exposure on the respiratory system and do not change the committee’s conclusions regarding the evidence of human health effects.

There is coherence across studies in humans, animals, and in vitro systems regarding the effect of e-cigarette exposure and respiratory symptoms. This adverse effect on respiratory symptoms is likely associated with an increase in cellular inflammation and oxidative stress and decreased cough reflexes and MCC. The observation that past e-cigarette use was associated with an increase in chronic bronchitic symptoms in adolescents and an increase in school absenteeism from asthma symptoms in current e-cigarette users is potentially concerning since a more rapid decline in lung function in later life has been linked to asthma and chronic bronchitis in early life (Bernal et al., 1989; Vestbo and Lange, 2016). In addition, there is limited evidence to indicate that e-cigarette substitution for tobacco product use in established smokers is associated with a decrease in cellular oxidative stress and improved respiratory symptoms and lung function.

Conclusion 11-1. There is no available evidence whether or not e-cigarettes cause respiratory diseases in humans.

Conclusion 11-2. There is limited evidence for improvement in lung function and respiratory symptoms among adult smokers with asthma who switch to e-cigarettes completely or in part (dual use).

Conclusion 11-3. There is limited evidence for reduction of chronic obstructive pulmonary disease (COPD) exacerbations among adult smokers with COPD who switch to e-cigarettes completely or in part (dual use).

Conclusion 11-4. There is moderate evidence for increased cough and wheeze in adolescents who use e-cigarettes and an association with e-cigarette use and an increase in asthma exacerbations.

Conclusion 11-5. There is limited evidence of adverse effects of e-cigarette exposure on the respiratory system from animal and in vitro studies.

VULNERABLE/SUSCEPTIBLE POPULATIONS

Chronic Obstructive Pulmonary Disease

Despite a number of studies, the results are unclear about whether use of e-cigarettes as a substitute for combustible tobacco use in people with COPD may be beneficial, neutral, or harmful. Harm may occur if e-cigarette use prevents the smoker from quitting entirely and instead prolongs the use of combustible tobacco products through dual use. Harm may also occur in an individual with COPD if single use of an e-cigarette as a substitute for combustible tobacco cigarettes causes additional airway inflammation in already damaged lungs. No studies have examined whether e-cigarette use alone can cause lower respiratory tract (LRT) inflammation in healthy adults or increase or decrease existing LRT inflammation in adults with COPD. If the use of e-cigarettes by a smoker with COPD can reduce use of combustible tobacco products and can decrease lung inflammation secondary to the reduction of exposure to toxicants found in combustible tobacco smoke but not e-cigarettes, this could be beneficial to the patients with COPD. In addition, individuals with COPD who failed or who are resistant to conventional NRT or other cessation strategies may be more willing to use e-cigarettes to quit smoking.

Asthma and Other Respiratory Diseases of Childhood

Asthma is one of the most common chronic respiratory diseases in the United States and is prevalent in young children and adolescents. In recent years since the introduction of e-cigarettes in the United States, substantial numbers of adolescents have tried and have used e-cigarettes (Backinger, 2017; HHS, 2016; Jamal et al., 2017; Kann et al., 2016; Miech et al., 2017).

Recent longitudinal studies have shown that children with asthma may have an accelerated decline in lung function as they age (Martinez, 2016). Smoking and other environmental exposures, including air pollution, can increase severity of asthma symptoms and these exposures have been associated with a more rapid decline in lung function in children (Gautier and Charpin, 2017; Schultz et al., 2017; Vanker et al., 2017). An area that remains unclear is if e-cigarette use can cause neutrophilic inflammation, similar to that which can be found in the asthmatic smoker and the smoker with COPD (Andelid et al., 2015; Siew et al., 2017). If so, then e-cigarette use by people with asthma may further exacerbate lower airway inflammation regardless of whether their asthma phenotype is predominately allergic or neutrophilic in origin. As discussed above, adolescents with asthma—a disease characterized by reversible airway obstruction—who use e-cigarettes may be more likely to have an increase in respiratory symptoms and exacerbations compared with adolescent non-users, as indicated by one cross-sectional study (Cho and Paik, 2016).

Cystic Fibrosis

Children and adolescents with other respiratory diseases who use e-cigarettes may also be at increased risk for worsening of respiratory symptoms. CF and primary ciliary dyskinesia (PCD) are respiratory diseases also characterized by lower respiratory tract neutrophilic inflammation. In the United States, the carrier frequency of CF mutations is 1/36, with whites having a carrier rate of 1/27 and African Americans with a carrier rate of 1/79 (Zvereff et al., 2014). It is unclear whether adolescents with CF or PCD would be more likely to try e-cigarettes if they perceive them to be less harmful than combustible tobacco cigarettes.

Nicotine alone has been shown to cause dysregulation of the CFTR chloride channel in the airways in animal studies, causing impaired airway MCC (Maouche et al., 2013). Another study found an association between secondhand smoke exposure in children with CF and lower FEV1 and weight percentile (Ong et al., 2017). Nicotine exposure from e-cigarette use could potentially cause a higher rate of respiratory symptoms in CF carriers if nicotine causes dysregulation of CFTR in the airways.

Preterm Infants

Exposure to nicotine in utero may have long-lasting negative effects on lung function in vulnerable populations such as preterm infants. Recently, maternal smoking during pregnancy has been associated with the development of bronchopulmonary dysplasia and later respiratory morbidities in preterm infants (Morrow et al., 2017). No studies have

examined the respiratory health effects of e-cigarettes on the preterm infants of mothers who used e-cigarettes during pregnancy.

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