Biomarkers of Exposure and Potential Harm Among Natural American Spirit Smokers

Home , Camel (cigarette), Pall Mall (cigarette), Reynolds American

Biomarkers of Exposure and Potential Harm among Natural American Spirit Smokers

Dana M. Carroll, PhD, MPH Astia Allenzara, MD Joni Jensen, MPH Irina Stepanov, PhD Stephen Hecht, PhD Sharon Murphy, PhD Xianghua Luo, PhD Eric Donny, PhD Dorothy K. Hatsukami, PhD

Objectives: We compared biomarkers of exposure and potential harm in smokers of American Spirit (AS) to smokers of Marlboro, Newport, Camel, and Pall Mall. Methods: We conducted secondary analysis on: (1) data from a randomized clinical trial (RCT); and (2) the Population As- sessment of Tobacco Use and Health (PATH) Study. Biomarkers analyzed included: total nicotine equivalents (TNE); 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL); N′-nitrosonornicotine and its N-glucuronide (total NNN);3-hydroxypropylmercapturic acid(3-HPMA); 2-hydroxypropylmercapturic acid (2-HPMA), 3-hydroxy-1 methylpropylmercapturic acid (HMPMA); S-phenylmercapturic acid(SPMA); 2-cyanoethylmercapturic acid (CEMA);

phenanthrene tetraol(PheT);1-hydroxypyrene (1-HOP);8-iso-PGF2α; white blood count(WBC); prostaglandin E metabolite(PGEM); and high sensitivity C-reactive protein(hsCRP). Results: AS smokers did not differ in TNE but had higher TNE per cigarette compared to other brands. Total NNAL, total NNN, CEMA, and 3-HPMA were lower in AS smokers. All other biomarkers were no different in AS smokers compared to all or the majority of the other brands.Conclusions : Levels of total NNAL, total NNN, acrylonitrile, and acrolein were reduced in AS smokers; however, it is not known whether reductions in exposure to these toxicants contribute to reduced harm. High- er TNE per cigarette smoked in AS smokers suggests a greater addictive potential compared to other brands. Regulatory action to ensure that consumers are not misled about the risks of the AS brand are recommended.

Key words: cigarette; disease risk; health risk; biological marker (biomarker); HPHC (harmful & potentially harmful constituents); tobacco exposure; tobacco marketing; tobacco regulation Tob Regul Sci.™ 2019;5(4):339-351 DOI: https://doi.org/10.18001/TRS.5.4.4

ultiple studies have shown that cigarette thermore, studies have shown youth to be partic- labels that use vocabulary such as “natu- ularly vulnerable to these descriptions and youth ral,” “organic,” and “additive-free” mis- do not notice or believe the mandatory warning Mlead consumers to perceive cigarettes with these statement: “Organic tobacco does not mean a safer descriptors to be less harmful than others.1-6 Fur- cigarette.”2,4,5 A study using data from the United

Dana M. Carroll, Postdoctoral Fellow, Masonic Cancer Center, University of Minnesota, Minneapolis, MN. Astia Allenzara, Resident, Hospital of the University of Pennsylvania, Philadelphia, PA. Joni Jensen, Research Manager, Masonic Cancer Center, University of Minnesota, Minneapolis, MN. Irina Stepanov, Associate Professor, Masonic Cancer Center, University of Minnesota, Minneapolis, MN. Stephen Hecht, Professor, Masonic Cancer Center, University of Minnesota, Minneapolis, MN. Sharon Murphy, Professor, Masonic Cancer Center, University of Minnesota, Minne- apolis, MN. Xianghua Luo, Associate Professor, Masonic Cancer Center, University of Minnesota, Minneapolis, MN. Eric C. Donny, Professor, Wake Forest School of Medicine, Winston-Salem, NC. Dorothy K. Hatsukami, Professor, Masonic Cancer Center, University of Minnesota, Minneapolis, MN. Correspondence Dr Carroll; [email protected]

Tob Regul Sci.™ 2019;5(4):339-351 339 Biomarkers of Exposure and Potential Harm among Natural American Spirit Smokers

States (US) representative Population Assessment breath alcohol level < .02%; smoked ≥ 5 cigarettes of Tobacco Use and Health (PATH) study found per day (CPD); an expired carbon monoxide level that smokers of the Natural American Spirit (AS) of > 8 ppm or if ≤ 8 ppm a urinary cotinine level of brand, which is a cigarette brand that includes de- > 1000 ng/mL. Exclusion criteria were the follow- scriptors like “additive-free,” “natural,” “organic,” ing: breastfeeding, pregnant or planning to become “tobacco and water,” as well as bright colors and pregnant; intentions to quit smoking in the next 30 American Indian imagery, were 23 times more like- days; used roll-your own cigarettes exclusively or to- ly to believe their brand was less harmful than oth- bacco products other than machine-manufactured er brands when compared with non-AS smokers.1 cigarettes for > 9 days of the past 30 days; had previ- The US Food and Drug Administration (FDA) ously used reduced nicotine content study cigarettes; has the regulatory authority to prohibit reduced unstable conditions or serious psychiatric or medical harm claims unless the product undergoes a rig- disease; and positive for illicit drug use (not includ- orous approval process as a modified risk tobacco ing cannabis). Participants were recruited between product (MRTP). In 2015, the FDA issued warn- July 2014 and September 2016. At baseline, par- ing letters to Santa Fe Natural Tobacco Com- ticipants provided a first morning void urine sample pany (SFNTC), manufacturer of the AS brand and a blood sample, and completed questionnaires and owned by Reynolds American, for labeling on demographics, medical, and smoking history. their cigarettes with the health-oriented market- As part of their smoking history, participants were ing terms.7 In 2017 an agreement was reached that asked: “What is your usual brand of cigarette (the resulted in SFNTC revising packaging to exclude type you smoke most often)?” Participants who re- “additive-free” and “natural” from marketing and ported AS, Marlboro, Newport, Camel or Pall Mall product labels; however, the use of “natural” in its as their usual brand were included in this analysis. brand name and the description “tobacco and wa- Additional information on the RCT methods and ter” are still allowed.8 procedures can be found elsewhere.13 The FDA has identified a list of 20 harmful or -po PATH is a nationally representative, longitudinal tentially harmful constituents (HPHC) in tobacco cohort study of adults and youth in the US, aged 12 products and tobacco smoke including tobacco- years and older.14 Wave 1 was conducted from Sep- specific nitrosamines (TSNAs), polycyclic aromatic tember 2013 to December 2014. Address-based, hydrocarbons (PAHs), volatile organic compounds area-probability sampling was used for recruitment. (VOCs), and nicotine.24 To date, no study has ex- Data are weighted so that estimates produced by amined exposure to HPHC or biomarkers of poten- the PATH Study are representative of the civilian, tial harm such as inflammation and oxidative stress noninstitutionalized US population. Additional in- in the actual users of AS cigarettes. Using baseline formation on the design and methods of the PATH data from a US 10-site randomized clinical trial study are available elsewhere.14 Data were restricted (RCT) and the PATH study, we compared a wide to adult daily cigarette smokers who provided urine range of tobacco-related biomarkers of exposure samples. Participants were asked: “Do you have a and potential harm in AS smokers with smokers regular brand of cigarettes that you usually smoke?” of Marlboro, Newport, Camel and Pall Mall brand If “yes,” participants were shown images with cig- cigarettes. We selected these particular brands for arette brand logos and asked to select their usual comparison since they represent the most popular brand. At the time of urine collection, participants cigarette brands among US young adults.11,12 were asked: “You said earlier that the regular brand of cigarettes you usually smoke is [usual brand]. METHODS Think about the very last cigarette you smoked. Was the brand your [usual brand]?” If “yes” and usual Secondary data analysis was conducted on 2 da- brand was AS, Marlboro, Newport, Camel or Pall tasets: (1) baseline data from a RCT of reduced Mall, participants were included in this analysis. nicotine content cigarettes among adult smokers conducted at 10 sites throughout the US and (2) the PATH Study. For the RCT, participants were eligible Biomarkers if they met the following criteria: ≥18 years of age; Table 1 provides a summary of the biomarkers

340 Carroll et al

Table 1 Biomarkers of Tobacco Exposure and Potential Harm Data FDA Constituent/Indication Biomarker Sample Source(s) HPHCa Nicotine Total nicotine equivalents (TNE)b Urine RCT; PATH Yes 4-(Methylnitrosamino)-1-(3- 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its Urine RCT; PATH Yes pyridyl)-1-butanone (NNK) glucuronides (total NNAL) N′-Nitrosonornicotine N′-nitrosonornicotine and its N-glucuronide (total Urine PATH Yes NNN) Acrolein 3-hydroxypropylmercapturic acid (3-HPMA) Urine RCT Yes Propylene oxide 2-hydroxypropylmercapturic acid (2-HPMA) Urine RCT Yes Benzene S-phenylmercapturic acid (SPMA) Urine RCT Yes Crotonaldehyde 3-hydroxy-1-methylpropylmercapturic acid (HMPMA) Urine RCT Yes Acrylonitrile 2-cyanoethylmercapturic acid (CEMA) Urine RCT Yes Phenanthrene Phenanthrene tetraol (PheT) Urine RCT No Pyrene Hydroxypyrene (1-HOP) Urine PATH No

Oxidative damage 8-iso-prostaglandin F2α (8-iso-PGF2α) Urine RCT NA Inflammation Prostaglandin E metabolite (PGEM) Urine RCT NA Inflammation High sensitivity C-reactive protein (hsCRP) Serum RCT NA Inflammation White blood cells (WBC) Blood RCT NA

Note. a: Included on the Food and Drug Administration list of harmful or potentially harmful constituents in tobacco or tobacco smoke24 b: In the RCT, TNE is the molar sum of total nicotine, total cotinine and total 3-hydroxycotinine, where “total” refers to the unconjugated and glucuronide conjugated forms. In PATH, TNE is the molar sum of total nicotine, total cotinine, total 3-hydroxycotinine, total cotinine N-oxide, total nicotine N-oxide, total norcotinine, and total nornicotine. measured and their data source. Assays of uri- 2 in the RCT. All urinary biomarkers values were nary biomarkers in the RCT were carried out in adjusted for creatinine to account for differences the Analytical Biochemistry Shared Resource of in urinary output among participants. Routine as- the Masonic Cancer Center, University of Min- says of the blood biomarkers, hsCRP and WBC, nesota as previously described for TNE (molar were conducted by the Advanced Research and sum of total nicotine, total cotinine and total Diagnostic Laboratory at the University of Min- 3-hydroxycotinine, where “total” refers to the un- nesota and Health East Medical Laboratories, St. conjugated and glucuronide conjugated forms),15 Paul, MN. total NNAL,16 PheT,16 3-HPMA,17 HMPMA,17 17 18 19 20 CEMA, 2-HPMA, SPMA, 8-iso-PGF2α, Data Analysis 21 and PGEM. Assays of TNE (molar sum of total Demographic and smoking history data were nicotine, total cotinine, total 3-hydroxycotinine, summarized across brands via means or propor- total cotinine N-oxide, total nicotine N-oxide, tions and 95% confidence intervals (CI). All bio- total norcotinine, and total nornicotine),22 total 23 23 24 markers had skewed distributions; therefore, they NNAL, total NNN, and 1-HOP in PATH were transformed using the natural logarithm to have been described previously. For biomarker val- approximate normality and were summarized us- ues below the limit of detection (LOD), the value ing geometric means (GM) and CI. Two linear was imputed as the LOD divided by the square regression models examining the relationship be- root of 2 in the PATH study and LOD divided by tween brand and each biomarker were employed:

Tob Regul Sci.™ 2019;5(4):339-351 DOI: https://doi.org/10.18001/TRS.5.4.4 341 Biomarkers of Exposure and Potential Harm among Natural American Spirit Smokers

Table 2 Demographics and Smoking History of Participants by Brand and Data Source RCT NAS Marlboro Newport Camel Pall Mall (N = 68) (N = 314) (N = 348) (N = 138) (N = 104) Age (years), mean 38.5 (35.1, 41.9) 41.8 (40.3, 43.3) 43.9 (42.7, 45.1)* 40.2 (38.0, 42.3) 52.6 (50.3, 54.9)* Male sex, % 57.4 (45.5, 68.4) 55.1 (49.6, 60.5) 54.9 (49.6, 60.0) 55.8 (47.5, 63.8) 57.7 (48.1, 66.7) > HS Education, % 85.3 (75.0, 91.8) 65.9 (60.5, 70.9)* 43.1 (38.0, 48.4)* 77.5 (69.9, 83.7) 66.4 (56.8, 74.7)* White race, % 80.3 (69.2, 88.1) 83.8 (79.3, 87.5) 21.4 (17.3, 26.0)* 82.4 (75.1, 87.8) 83.5 (75.1, 89.4) Body mass index, mean 26.2 (24.9, 27.4) 28.9 (28.2, 29.7)* 31.2 (30.4, 32.0)* 29.4 (28.1, 30.6)* 29.0 (27.8, 30.1)* Years smoking, mean 20.5 (17.1, 23.9) 24.7 (23.2, 26.1) 24.9 (23.7, 26.2) 22.6 (20.4, 24.8) 33.9 (31.3, 36.6)* CPD, mean 12.6 (11.3, 13.9) 19.1 (18.1, 20.0)* 15.5 (14.7, 16.4)* 18.1 (16.7, 19.5)* 20.3 (18.6, 22.1)* TNE, GM 58.9 (49.8, 69.6) 58.8 (54.8, 63.0) 48.3 (45.1, 51.8) 59.5 (53.6, 66.1) 73.1 (66.8, 79.9) TNE/CPD, GM 5.1 (4.4, 5.9) 3.4 (3.2, 3.6)* 3.5 (3.2, 3.8)* 3.6 (3.3, 4.0)* 4.0 (3.6, 4.4) PATHa NAS Marlboro Newport Camel Pall Mall (N = 392,143) (N = 9,256,656) (N = 4,428,971) (N = 1,921,109) (N = 1,970,381) Age (years), mean 50.5 (40.8, 60.1) 39.3 (38.1, 40.5) 37.0 (35.2, 38.9)* 34.7 (33.1, 36.3)* 50.1 (46.6, 53.6) Male sex, % 48.1 (24.6, 72.5) 55.5 (51.1, 59.9) 58.1 (51.9, 64.1) 49.9 (39.9, 59.9) 48.6 (41.4, 55.9) > HS Education, % 65.2 (31.8, 88.3) 48.3 (43.6, 53.0) 31.8 (25.7, 38.6) 56.6 (47.8, 65.0) 31.7 (25.9, 38.1) White race, % 90.7 (74.8, 97.0) 84.7 (81.7, 87.2) 31.6 (22.0, 43.0)* 76.8 (65.4, 85.3) 86.8 (81.5, 90.8) Body mass index, mean 23.8 (22.2, 25.5) 27.4 (26.7, 28.0)* 29.2 (28.0, 30.4)* 28.2 (26.7, 29.7)* 28.2 (27.0, 29.5)* Years smoking, mean 30.7 (18.5, 42.8) 20.7 (19.6, 21.8) 17.3 (15.9 , 18.8) 15.5 (14.1, 17.0) 31.7 (29.0, 34.4) CPD, mean 14.3 (10.4, 18.3) 16.0 (15.3, 16.7) 13.6 (12.1, 15.1) 13.3 (11.8, 14.7) 17.0 (15.6, 18.5) TNE, GM 97.4 (53.2, 178.5) 65.9 (61.0, 71.2) 50.0 (43.6, 57.4) 51.1 (46.1, 56.7) 85.0 (76.6, 94.3) TNE/CPD, GM 8.5 (6.1, 11.8) 4.8 (4.5, 5.1)* 4.3 (4.0, 4.7)* 4.5 (4.0, 5.1)* 5.7 (5.2, 6.2)

Note. * Indicates a statistically significance difference (p < .013) from the American Spirit (AS) brand. a: Population Assessment of Tobacco Use and Health (PATH) data is weighted. RCT: Randomized controlled trial data GM: Geometric mean Parentheses include 95% confidence intervals CPD: Cigarettes per day TNE: total nicotine equivalents HS: high school

(1) unadjusted; and (2) adjusted for age, sex, race, parison between AS and each of the other cigarette body mass index (BMI), TNE (log-transformed), brands. Analyses were carried out in SAS Version and duration of smoking fairly regularly (calculat- 9.4 (SAS Institute Inc, Cary, NC). Additionally, ed in the RCT as ‘age’ - ‘age started smoking fairly all analyses using PATH data were performed us- regularly’; defined in the PATH as ‘time smoking ing survey procedures (eg, PROC SURVEYFREQ, fairly regularly’). In the adjusted analysis of the PROC SURVEYREG) and weights that adjust for PATH data, time since last cigarette also was in- the complex sampling design. PATH data variances cluded because the urine sample was a spot and not were estimated by the balanced repeated replica- a first morning void.An alpha of .013 for statisti- tion method26 with Fay’s adjustment set to 0.3 to cal significance was used to account for the com- increase estimate stability.27

342 Carroll et al

Figure 1 Geometric Means and 95% Confidence Intervals of Urinary Biomarkers of Tobacco-related Exposure by Brand

A B 2 3.5 * 3

1.5 * * 2.5

* * * * 2 * 1 * 1.5

1 0.5 0.5 * * * * 0 0 RCT NNAL PATH NNAL PATH NNN RCT Phet PATH 1-HOP (pmol/mg) (pmol/mg) (pmol/mg) (pmol/mg) (pmol/mg) C 9 * 8 * * 7 6 * 5 4

3 2 * * * 1 0 RCT 3-HPMA RCT 2-HPMA RCT HMPMA RCT SPMA RCT CEMA (nmol/mg) (nmol/mg) (nmol/mg) (pmol/mg) (nmol/mg)

American Spirit Marlboro Newport Camel Pall Mall

Note. A: tobacco-specific nitrosamines by brand B: polycyclic aromatic hydrocarbons (PAH) by brand C: mercapturic acids by brand Data from the randomized clinical trial (RCT) or the Population Assessment of Tobacco Use and Health (PATH) study. All estimates are adjusted for age, sex, race, body mass index, total nicotine equivalents (log-transformed), a and duration regular smoking. PATH estimates include an additional adjustment for time since last cigarette * Indicates a statistically significance difference (p < .013) from AS brand.

Tob Regul Sci.™ 2019;5(4):339-351 DOI: https://doi.org/10.18001/TRS.5.4.4 343 Biomarkers of Exposure and Potential Harm among Natural American Spirit Smokers

Figure 2 Geometric Means and 95% Confidence Intervals of Urinary and Blood Biomarkers of Potential Harm by Brand

9 8 7 6 5 4 3 2 * 1 0 8-isoPGF2α hsCRP WBC PGEM (pmol/mg) (mg/L) (1000/µL) ([pmol/mg]/10)

American Spirit Marlboro Newport Camel Pall Mall

Note. Data from the randomized clinical trial (RCT). All estimates are adjusted for age, sex, race, body mass index, total nicotine equivalents (log-transformed), and duration regular smoking. * Indicates a statistically significance difference (p < .013) from AS brand.

RESULTS pared to the other brands; CPD in the US adult In the PATH study, unweighted sample sizes for population did not differ between AS smokers and each of the cigarette brands were as follows: N = smokers of the other brands. In both the RCT and 43 for AS, N = 1233 for Marlboro, N = 636 for US adult population, AS smokers did not differ Newport, N = 318 for Camel, and N = 240 for from other brand smokers in levels of TNE, but Pall Mall. All PATH data presented in the tables had higher TNE per cigarette smoked. and figures and described hereafter are weighted to reflect the US adult population. Table 2 presents Tobacco-Specific Nitrosamines (TSNA) characteristics of the RCT and PATH samples by Unadjusted and adjusted geometric means are brand. In the RCT, AS smokers were younger on presented in Supplementary Tables 1-2. Medians average than other brand smokers, and AS smokers and interquartile ranges are presented in Supple- in the US adult population were older than other mentary Table 3. The results of the adjusted anal- brand smokers. Reflecting their age, AS smokers yses examining TSNA by brand are presented in in the RCT had a shorter duration of smoking Figure 1 (A). In both the RCT and the US adult than smokers of other brands; US adult AS smok- population, the adjusted GM value of total NNAL ers trended toward having a longer smoking dura- was lower in AS smokers compared to smokers of tion. In both the RCT and US adult population, the other brands. In the US adult population, the AS smokers were more educated, although this was adjusted GM value of total NNN was lower in AS not statistically significant in the US adult popula- smokers compared to smokers of other popular tion. AS smokers in the RCT had lower CPD com- brands.

344 Carroll et al

Polycyclic Aromatic Hydrocarbons (PAH) the mercapturic acids crotonaldehyde, propylene Figure 1 (B) presents results of the adjusted oxide, and benzene. analyses examining PAH. In the RCT, PheT was Tobacco-specific nitrosamines NNK and NNN significantly higher in AS smokers versus Newport are potent carcinogens of the lung and esophagus, smokers, but this difference was not observed when respectively, based on carcinogenicity studies in AS was compared to any of the other brand smok- laboratory animals.28 In our study, levels of their ers. In the US adult population, 1-HOP levels were corresponding urinary biomarkers total NNAL and no different in AS compared to smokers of other total NNN were 3 times higher in smokers of other brands. cigarette brands compared to AS smokers. Previous analyses of these biomarkers in smokers from the Mercapturic Acids Shanghai Cohort Study (SCS) have documented a Figure 1 (C) presents results of the adjusted statistically significant relationship between urinary total NNAL and lung cancer and between urinary analyses examining mercapturic acids in the RCT. 29-31 Levels of 3-HPMA and CEMA were lower in AS total NNN and esophageal cancer in humans. smokers compared to smokers of the other brands, Although lower than other brands, urinary total NNAL and NNN present in AS smokers were at except Newport. HMPMA was lower in AS smok- levels that have been associated with increases in ers compared to Pall Mall smokers, but this differ- 29-31 ence was not observed for any of the other brand lung and esophageal cancer risks. Addition- smokers. Levels of 2-HPMA and SPMA were no ally, co-exposure with other toxicants present in different in AS smokers compared to smokers of AS cigarettes may enhance carcinogenicity. Thus, the other brands. it is unclear whether the reductions in NNAL and NNN observed among AS smokers correspond to a clinically meaningful decrease in cancer risk. Im- Inflammation and Oxidative Stress portantly, the tobacco plant itself and its processing Figure 2 presents results of the adjusted analyses technique determine the levels of these important examining biomarkers of oxidative stress and in- carcinogens in cigarette smoke.32 Therefore, lower flammation by brand in the RCT. AS smokers had levels of NNK and NNN exposure in smokers of lower levels of 8-isoPGF2α than Newport smok- AS cigarettes should not be attributed to the lack ers, but this difference was not observed for any of of additives or due to tobacco being “natural” or the other brand smokers. For hsCRP, WBC, and “organic.” PGEM, there was no difference between AS smok- Nearly everyone is exposed environmentally or ers and smokers of the other brands. endogenously to acrolein, crotonaldehyde, propylene oxide, benzene, and acrylonitrile; therefore, the DISCUSSION mercapturic acid metabolites of these compounds This is the first study to provide an analysis of are found in essentially all human urine. Levels are tobacco-related exposure and potential harm bio- frequently higher in smokers than in nonsmokers 33 markers in smokers of the AS brand, a cigarette and decrease quickly upon smoking cessation. brand viewed by consumers as less harmful than CEMA, a metabolite of the carcinogen and respira- other commercial cigarette brands.1-6 We utilized tory toxicant acrylonitrile, and 3-HPMA, a metab- 2 data sources and demonstrated that AS smokers olite of the respiratory and cardiovascular toxicant had lower levels of exposure to biomarkers of to- acrolein, were lower in AS smokers compared to bacco-specific nitrosamines and mercapturic acids the other brands except for Newport. Levels of of acrylonitrile and acrolein than smokers of other HMPMA, a metabolite of the carcinogen croton- popular cigarette brands. Our results also showed aldehyde, 2-HPMA, a metabolite of the carcino- that AS smokers had higher nicotine exposure gen and respiratory toxicant propylene oxide, and per cigarette smoked and they did not differ from SPMA, an established biomarker of the carcinogen, smokers of the other brands in levels of inflam- cardiovascular toxicant, and reproductive toxicant matory and oxidative stress biomarkers, polycyclic benzene, in AS were no different than the other aromatic hydrocarbon markers, and biomarkers of brands. These results suggest that levels of acrylo-

Tob Regul Sci.™ 2019;5(4):339-351 DOI: https://doi.org/10.18001/TRS.5.4.4 345 Biomarkers of Exposure and Potential Harm among Natural American Spirit Smokers nitrile and acrolein are likely lower in the AS ciga- Study limitations include the lack of assessing AS rettes compared to the other cigarette brands; there varieties (total of 10) which, as Jain et al show,45 is no difference in levels of crotonaldehyde, propyl- vary in constituent levels and the cross-sectional ene oxide, and benzene in AS cigarettes compared nature of the data that impedes causal interpreta- to the other brands. tion. Whereas the goal was to compare biomarkers Polycyclic aromatic hydrocarbon exposure re- among exclusive users of the AS brand to exclusive sults from incomplete combustion of organic mat- users of the other cigarette brands, it is possible ter, including cigarettes.34 Whereas phenanthrene that use of other tobacco products or marijuana and pyrene are not carcinogenic themselves, they may have impacted the results. Exclusion criteria are part of a mixture of PAH, many of which are for the RCT include use of other tobacco prod- carcinogens.34 Phenanthrene tetraol (PheT), a me- ucts > 9 times per month; thus, the RCT did not tabolite of the phenanthrene, and 1-HOP, a me- include frequent users of other tobacco products. tabolite of pyrene, are widely employed biomarkers Data from the PATH study were not restricted of PAH exposure. Given that PAHs are ubiquitous based on use of other tobacco products. Thus, we environmental and dietary contaminants, nearly examined someday or everyday use of other to- all humans have PheT and 1-HOP in their urine; bacco products and this represented 29% of the however, levels are generally higher in smokers than participants from the PATH study included in this in nonsmokers. Levels of both PHeT and 1-HOP analysis (27% of the AS smokers). In a sensitivity in AS smokers were no different than levels ob- analysis excluding these individuals, we observed served in the other cigarette brands. similar results for each of the biomarkers examined There are multiple pathways from tobacco ex- (total NNAL, NNN, and 1-Hop) to our results posure to the development of disease, such as when including them. Specifically, total NNAL cardiovascular disease and cancer, which include and NNN remained significantly lower among AS inflammation and oxidative stress.35 Widely used smokers compared to the other brands; 1-Hop did biomarkers of inflammation and oxidative stress not differ. Another limitation of the study is the use of biomarkers that are not specific to tobacco include WBC, hsCRP, PGEM, and 8-iso-PGF2α. Levels of WBC,36-38 hsCRP,39,40 PGEM,41,42 and use, with the exception of total NNAL and NNN; 41 therefore, results may reflect environmental and/or 8-iso-PGF2α are generally higher in smokers than nonsmokers and decrease following cessation. In endogenous exposures. our study, levels of these inflammatory and oxida- Strengths of this study include the use of data tive stress biomarkers in AS smokers did not differ from 2 separate studies that differed in many as- from levels in smokers of other cigarette brands. pects (eg, study design, eligibility criteria, recruit- American Spirit brand smokers did not differ ment methods, measures available to identify AS from other brand smokers in levels of total nico- brand smokers). Differences in study design and tine exposure, but had higher nicotine per cigarette methods of recruitment likely resulted in the ob- smoked. Although it is plausible that AS smok- served differences in the demographics and smokers have different smoking topography (eg smoke ing history of AS smokers in the 2 studies. For more intensely), it is also likely that AS cigarettes example, AS smokers were, on average, 38.5 years contain more nicotine as previous studies have in the RCT compared to 50.5 years in the PATH shown variation in nicotine levels per cigarette study. Despite these differences, patterns in the bio- across different brands.43 Research by Jain et al45 markers measured in both studies (ie, TNE, TNE included in this special issue showed that levels of per each cigarette smoked, and total NNAL) were nicotine are higher in AS cigarettes compared to remarkably similar and demonstrate consistency of other commercial cigarette brands. These findings the results. Additional strengths of this study in- suggest that AS cigarettes may have a greater ad- clude the use of popular US cigarette brands for dictive potential than other cigarette brands. This comparison to the AS brand and measurement of is particularly concerning given that the AS brand both exposure and effect biomarkers descriptors such as “natural,” “organic,” and “addi- In conclusion, although levels of total NNAL tive-free” are appealing to youth.2,4,5 and NNN, and biomarkers of acrylonitrile and ac-

346 Carroll et al rolein were reduced in AS smokers, it is not known the health risks of the AS brand, regulatory efforts whether these reductions contribute to reduced to restrict AS marketing from misleading consum- harm compared to other cigarette brands, particu- ers, as well as educational campaigns and policy ac- larly given that levels of crotonaldehyde, propylene tion tailored to young adults are recommended. oxide, benzene, polycyclic aromatic hydrocarbons, inflammation, and oxidative stress biomarkers in Human Subjects Statement AS smokers did not differ from smokers of all or The University of Minnesota institutional review the majority of the other brands studied. However, board reviewed and approved this study. it is clear that AS smokers are still exposed to multiple toxicants and carcinogens that are integral in the development of tobacco-related disease. Addi- Conflict of Interest Statement tionally, AS brand smokers had higher nicotine per None declared. cigarette smoked which suggests a greater addictive potential of this brand. Given these results, we rec- Acknowledgements ommend regulatory action to ensure that consum- Funding was provided by the National Institutes ers are not misled about the health risks of the AS of Health Award U54 DA031659 from the Na- brand. tional Institute on Drug Abuse and P01CA217806 from the Natioinal Cancer Institute. This investi- IMPLICATIONS FOR TOBACCO gation was also supported by National Institutes REGULATION of Health, National Research Service Award T32 Our results have implications for the US FDA DA007097 from the National Institute on Drug Center for Tobacco Products and tobacco regula- Abuse. tory agencies worldwide. In the US, AS cigarettes are the fifth most popular brand among young References 12 adults. The US market share of AS cigarettes rose 1. Pearson JL, Johnson A, Villanti A, et al. Misperceptions from 0.26% in 2002 to 1.70% in 2013.11 This in- of harm among Natural American Spirit smokers: results crease in market share and popularity with young from wave 1 of the Population Assessment of Tobacco and Health (PATH) study (2013–2014). Tob Control. adults may be due to the brand’s deceptive pack- 2017;26(e1):e61-e67. aging and advertising. The US FDA’s 2015 state- 2. Czoli CD, Hammond D. Cigarette packaging: youth ment to SFNTC said: “Your product labeling for perceptions of “Natural” cigarettes, filter references, and Natural American Spirit cigarettes, which uses the contraband tobacco. J Adolesc Health. 2014;54(1):33-39. descriptors “natural” and “additive free,” represents 3. O’Connor RJ, Lewis MJ, Adkison SE, et al. Perceptions of “Natural” and “Additive-Free” cigarettes and intentions explicitly and/or implicitly that the products or to purchase. Health Educ Behav. 2016;44(2):222-226. their smoke do not contain or are free of a sub- 4. Arnett JJ. Winston’s “No Additives” campaign: “straight stance and/or that the products present a lower risk up”? “no bull”? Public Health Rep. 1999;114(6):522-527. of tobacco-related disease or are less harmful than 5. Byron MJ, Baig SA, Moracco KE, Brewer NT. Adoles- cents’ and adults’ perceptions of ‘natural’, ‘organic’ and one or more other commercially marketed tobacco ‘additive-free’ cigarettes, and the required disclaimers. Tob 7 products.” This study does not provide support Control. 2016;25(5):517-520. for an assumption that descriptors such as “natu- 6. Pearson JL, Richardson A, Feirman SP, et al. American ral” and “addictive free” translate to a reduction in Spirit pack descriptors and perceptions of harm: a crowd- sourced comparison of modified packs. Nicotine Tob Res. harm. Worldwide nearly 200 countries, which be- 2016;18(8):1749-1756. long to the World Health Organization Framework 7. US Food and Drug Administration, Center for Tobacco Convention on Tobacco Control, have committed Products. Warning letter to Santa Fe Natural Tobacco to banning any tobacco product packaging and la- Company, Inc. 8/27/15. 2015. Available at: https://www. fda.gov/ICECI/EnforcementActions/%20WarningLet- belling that “…promote a tobacco product by any ters/2015/ucm459778.htm. Accessed July 5, 2018. means that are false, misleading, deceptive or likely 8. Epperson AE, Henriksen L, Prochaska JJ. Natural Ameri- to create an erroneous impression about its charac- can Spirit brand marketing casts health halo around teristics, health effects, hazards or emissions.”44 To smoking. Am J Public Health. 2017;107(5):668-670. 9. Iffland Y, Müller R, Groneberg D, Gerber A. High partic- ensure that consumers are not further misled about ulate matter emission from additive-free Natural Ameri-

Tob Regul Sci.™ 2019;5(4):339-351 DOI: https://doi.org/10.18001/TRS.5.4.4 347 Biomarkers of Exposure and Potential Harm among Natural American Spirit Smokers

can Spirit cigarettes. Springerplus. 2016;5(1):1958. vices, National Institutes of Health, National Institute 10. Cecil TL, Brewer TM, Young M, Holman MR. Acro- on Drug Abuse, and US Food and Drug Administration lein yields in mainstream smoke from commercial ciga- Center for Tobacco Products. Population Assessment of rette and little cigar tobacco products. Nicotine Tob Res. Tobacco and Health (PATH) Study [United States] re- 2017;19(7):865-870. stricted-use files. Lab procedures manual for urine panel 11. Sharma A, Fix BV, Delnevo C, et al. Trends in market tobacco-specific nitrosamines (TSNA). Ann Arbor, MI: share of leading cigarette brands in the USA: national Inter-university Consortium for Political and Social Re- survey on drug use and health 2002–2013. BMJ Open. search; 2018. 2016;6(1):e008813. 24. United States Department of Health and Human Ser- 12. Glasser AM, Johnson AL, Rath JM, et al. Tobacco prod- vices, National Institutes of Health, National Institute uct brand preference among US Young adults, 2011- on Drug Abuse, and US Food and Drug Administration 2014. Tob Regul Sci. 2016;2(1):44-55. Center for Tobacco Products. Population Assessment of 13. Hatsukami DK, Luo X, Jensen JA, et al. Effect of im- Tobacco and Health (PATH) Study [United States] re- mediate vs gradual reduction in nicotine content of ciga- stricted-use files. Lab procedures manual for urine panel rettes on biomarkers of smoke exposure: a randomized hydroxy polycyclic aromatic hydrocarbons (PAH). Ann clinical trial. JAMA. 2018;320(9):880-891. Arbor, MIC: Inter-university Consortium for Political 14. Hyland A, Ambrose BK, Conway KP, et al. Design and and Social Research; 2018. methods of the Population Assessment of Tobacco and 25. US Food and Drug Administration. Harmful and Po- Health (PATH) Study. Tob Control. 2017;26(4):371-378. tentially Harmful Constituents in Tobacco Products and 15. Murphy SE, Villalta P, Ho SW, von Weymarn LB. Anal- Tobacco Smoke: Established List. 2012. Available at: ysis of [3’,3’-d(2)]-nicotine and [3’,3’-d(2)]-cotinine https://www.fda.gov/TobaccoProducts/Labeling/Rules- by capillary liquid chromatography-electrospray tan- RegulationsGuidance/ucm297786.htm. Accessed July dem mass spectrometry. J Chromatogr B Analyt Technol 13, 2018. Biomed Life Sci. 2007;857(1):1-8. 26. McCarthy PJ. Pseudoreplication: further evaluation and 16. Carmella SG, Ming X, Olvera N, et al. High throughput applications of the balanced half-sample technique. Vital liquid and gas chromatography-tandem mass spectrom- Health Stat 2. 1969 Jan;(31):1-24. etry assays for tobacco-specific nitrosamine and polycyclic 27. Judkins DR. Fay’s method for variance estimation. J Off aromatic hydrocarbon metabolites associated with lung Stat. 1990;6(3):223-239. cancer in smokers. Chem Res Toxicol. 2013;26(8):1209- 28. International Agency for Research on Cancer (IARC). 1217. Smokeless Tobacco and Some Tobacco-Specific N-Nitrosa- 17. Carmella SG, Chen M, Zarth A, Hecht SS. High through- mines. Lyon, France: IARC; 2007. put liquid chromatography-tandem mass spectrometry 29. Yuan J-M, Gao Y-T, Murphy SE, et al. Urinary levels of assay for mercapturic acids of acrolein and crotonalde- cigarette smoke constituent metabolites are prospectively hyde in cigarette smokers’ urine. J Chromatogr B Analyt associated with lung cancer development in smokers. Technol Biomed Life Sci. 2013;935:36-40. Cancer Res. 2011;71(21):6749-6757. 18. Zarth AT, Carmella SG, Le CT, Hecht SS. Effect of 30. Stepanov I, Sebero E, Wang R, et al. Tobacco-specific N- cigarette smoking on urinary 2-hydroxypropylmer- nitrosamine exposures and cancer risk in the Shanghai capturic acid, a metabolite of propylene oxide. J Chro- Cohort Study: remarkable coherence with rat tumor sites. matogr B Analyt Technol Biomed Life Sci. 2014;953- Int J Cancer. 2014;134(10):2278-2283. 954:126-131. 31. Yuan J-M, Knezevich AD, Wang R, et al. Urinary levels 19. Carmella SG, Chen M, Han S, et al. Effects of smoking of the tobacco-specific carcinogenN′ -nitrosonornicotine cessation on eight urinary tobacco carcinogen and toxi- and its glucuronide are strongly associated with esophageal cant biomarkers. Chem Res Toxicol. 2009;22(4):734-741. cancer risk in smokers. Carcinogenesis. 2011;32(9):1366- 20. Yan W, Byrd GD, Ogden MW. Quantitation of isopros- 1371. tane isomers in human urine from smokers and nonsmok- 32. Hecht SS. It is time to regulate carcinogenic tobacco- ers by LC-MS/MS. J Lipid Res. 2007;48(7):1607-1617. specific nitrosamines in cigarette tobacco. Cancer Prev Res 21. Neale JR, Dean BJ. Liquid chromatography-tandem mass (Phila). 2014;7(7):639-647. spectrometric quantification of the dehydration product 33. Hecht SS, Carmella SG, Kotandeniya D, et al. Evaluation of tetranor PGE-M, the major urinary metabolite of pros- of toxicant and carcinogen metabolites in the urine of e- taglandin E(2) in human urine. J Chromatogr B Analyt cigarette users versus cigarette smokers. Nicotine Tob Res. Technol Biomed Life Sci. 2008;871(1):72-77. 2015;17(6):704-709. 22. United States Department of Health and Human Services 34. International Agency for Research on Cancer (IARC). (USDHHS), National Institutes of Health, National In- Some non-heterocyclic polycyclic aromatic hydrocar- stitute on Drug Abuse, and US Food and Drug Admin- bons and some related exposures. IARC Monogr Eval istration Center for Tobacco Products. Population As- Carcinog Risks Hum. 2010;92:1-853. sessment of Tobacco and Health (PATH) Study [United 35. US Department of Health and Human Services. The States] restricted-use files. Lab procedures manual for Health Consequences of Smoking—50 years of Progress: A Urine Panel - Urinary Nicotine Metabolites (Cotinine Report of the Surgeon General. 2014. Available at: https:// and Hydroxycotinine) (UNICM). Ann Arbor, MI: Inter- www.surgeongeneral.gov/library/reports/50-years-of- university Consortium for Political and Social Research; progress/index.html. Accessed April 28, 2019. 2018. 36. Smith MR, Kinmonth AL, Luben RN, et al. Smok- 23. United States Department of Health and Human Ser- ing status and differential white cell count in men and

348 Carroll et al

women in the EPIC-Norfolk population. Atherosclerosis. ceptibility in smokers. J Thorac Oncol.2016;11(2):S7-S8. 2003;169(2):331-337. 42. Duffield-Lillico AJ, Boyle JO, Zhou XK, et al. Levels of 37. Grimm RH, Jr, Neaton JD, Ludwig W. Prognostic im- Prostaglandin E metabolite and Leukotriene E(4) are in- portance of the white blood cell count for coronary, can- creased in the urine of smokers. Evidence that Celecoxib cer, and all-cause mortality. JAMA. 1985;254(14):1932- shunts arachidonic acid into the 5-Lipoxygenase pathway. 1937. Cancer Prev Res (Phila). 2009;2(4):322-329. 38. Abel GA, Hays JT, Decker PA, et al. Effects of biochemi- 43. Calafat AM, Polzin GM, Saylor J, et al. Determination cally confirmed smoking cessation on white blood cell of tar, nicotine, and carbon monoxide yields in the main- count. Mayo Clin Proc. 2005;80(8):1022-1028. stream smoke of selected international cigarettes. Tob 39. Bazzano LA, He J, Muntner P, et al. Relationship be- Control. 2004;13(1):45-51. tween cigarette smoking and novel risk factors for car- 44. World Health Organization. Tobacco Free Initiative diovascular disease in the United States. Ann Intern Med. (TFI): Article 11 of the WHO Framework Convention 2003;138(11):891-897. on Tobacco Control - Packaging and labelling of tobacco 40. Ohsawa M, Okayama A, Nakamura M, et al. CRP levels products. Available at: http://www.who.int/tobacco/in- are elevated in smokers but unrelated to the number of dustry/product_regulation/art_11_fctc/en/ . Accessed cigarettes and are decreased by long-term smoking ces- July 5, 2018. sation in male smokers. Prev Med. 2005;41(2):651-656. 45. Jain V, Aleksandra A, Darlene H, et al. Comprehensive 41. Hecht SS, Carmella SG, Murphy SE, et al. Tobacco smoke chemical characterization of Natural American Spirit toxicant and carcinogen biomarkers and lung cancer sus- packs Tob Regul Sci. 2019:5(4):381-399.

Tob Regul Sci.™ 2019;5(4):339-351 DOI: https://doi.org/10.18001/TRS.5.4.4 349 Biomarkers of Exposure and Potential Harm among Natural American Spirit Smokers

Supplementary Table 1 Unadjusted and Adjusted Geometric Mean Values of Biomarkers by Brand in the RCT RCT

AS Marlboro Newport Camel Pall Mall (N = 68) (N = 314) (N = 348) (N = 38) (N = 104)

Total NNAL pmol/mga 0.35 (0.28, 0.44) 1.74 (1.60, 1.90)* 0.99 (0.92, 1.07)* 0.99 (0.85, 1.15)* 1.67 (1.47, 1.89)*

Total NNAL pmol/mgb 0.37 (0.30, 0.45) 1.71 (1.58, 1.84) * 1.12 (1.05, 1.20) * 0.97 (0.86, 1.09) * 1.23 (1.10, 1.38) *

3-HPMA nmol/mga 5.57 (4.73, 6.56) 6.89 (6.39, 7.43) 5.62 (5.23, 6.03) 7.20 (6.42, 8.07)* 9.07 (7.85, 10.48)*

3-HPMA nmol/mgb 5.65 (5.00, 6.38) 6.89 (6.47, 7.34)* 6.09 (5.71, 6.49) 7.17 (6.50, 7.92)* 7.52 (6.70, 8.43)*

2-HPMA nmol/mga 0.68 (0.56, 0.82) 0.71 (0.65, 0.78) 0.60 (0.55, 0.65) 0.80 (0.71, 0.91) 0.77 (0.66, 0.90)

2-HPMA nmol/mgb 0.69 (0.56, 0.85) 0.72 (0.65, 0.79) 0.62 (0.56, 0.67) 0.79 (0.70, 0.90) 0.71 (0.60, 0.83)

HMPMA nmol/mga 3.41 (2.94, 3.97) 3.99 (3.72, 4.28) 3.26 (3.04, 3.51) 3.93 (3.51, 4.39) 5.57 (4.85, 6.40)*

HMPMA nmol/mgb 3.44 (3.07, 3.86) 4.01 (3.79, 4.23) 3.54 (3.33, 3.77) 3.92 (3.60, 4.28) 4.60 (4.12, 5.13)*

CEMA nmol/mga 0.51 (0.41, 0.62) 0.69 (0.63, 0.75)* 0.53 (0.49, 0.58) 0.70 (0.61, 0.81)* 0.91 (0.79, 1.04)*

CEMA nmol/mgb 0.49 (0.43, 0.57) 0.68 (0.64, 0.72)* 0.60 (0.56, 0.64) 0.69 (0.62, 0.76)* 0.73 (0.65, 0.81)*

SPMA pmol/mga 3.08 (2.33, 4.08) 3.54 (3.14, 3.99) 2.91 (2.63, 3.22) 3.98 (3.36, 4.71) 3.75 (3.01, 4.68)

SPMA pmol/mgb 3.15 (2.53, 3.91) 3.32 (2.99, 3.67) 3.43 (3.12, 3.77) 3.69 (3.18, 4.30) 2.85 (2.31, 3.53)

PheT pmol/mga 2.87 (2.40, 3.44) 2.44 (2.26, 2.64) 1.73 (1.59, 1.87)* 2.61 (2.30, 2.95) 2.43 (2.13, 2.78)

PheT pmol/mgb 2.63 (2.21, 3.12) 2.18 (2.01, 2.37) 2.01 (1.85, 2.18)* 2.34 (2.07, 2.64) 2.00 (1.74, 2.30)

8-isoPGF2α pmol/mga 1.03 (0.88, 1.21) 1.17 (1.08, 1.26) 1.30 (1.21, 1.41)* 1.25 (1.10, 1.43) 1.09 (0.92, 1.28)

8-isoPGF2α pmol/mgb 0.95 (0.81, 1.11) 1.11 (1.02, 1.21) 1.30 (1.20, 1.41)* 1.18 (1.02, 1.35) 1.16 (0.98, 1.37)

PGEM pmol/mga 44.66 (35.47, 56.23) 50.21 (44.73, 56.36) 60.30 (53.90, 67.46) 45.96 (38.38, 55.03) 45.57 (36.31, 57.20)

PGEM pmol/mgb 40.73 (32.60, 50.88) 49.55 (43.93, 55.88) 56.66 (50.12, 64.06) 45.34 (37.66, 54.57) 47.26 (37.12, 60.16)

hsCRP mg/La 1.52 (1.15, 2.00) 2.03 (1.82, 2.27) 2.44 (2.14, 2.77)* 1.98 (1.65, 2.38) 2.46 (1.99, 3.05)*

hsCRP mg/Lb 2.01 (1.55, 2.61) 2.11 (1.89, 2.35) 2.23 (1.97, 2.52) 2.07 (1.77, 2.44) 2.18 (1.78, 2.67)

WBC 103/µLa 6.96 (6.54, 7.40) 7.70 (7.49, 7.92)* 6.66 (6.45, 6.89) 7.41 (7.07, 7.77) 7.42 (7.04, 7.82)

WBC 103/µLb 6.90 (6.46, 7.37) 7.43 (7.20, 7.66) 6.86 (6.63, 7.09) 7.11 (6.77, 7.47) 7.11 (6.74, 7.47)

Note. Data from a randomized clinical trial (RCT) * indicates a statistically significance difference (p < .013) from American Spirit (AS) brand Parentheses include 95% confidence intervals a: Unadjusted value b: Adjusted for age, sex, race, body mass index, total nicotine equivalents (log-transformed), and duration regular smoking.

350 Carroll et al

Supplementary Table 2 Unadjusted and Adjusted Geometric Mean Values of Biomarkers by Brand in the PATH Study PATHa

AS Marlboro Newport Camel Pall Mall (N = 392,143) (N = 9,256,656) (N = 4,428,971) (N = 1,921,109) (N = 1,970,381)

Total NNAL pmol/mgb 0.47 (0.27, 0.83) 1.56 (1.44, 1.70)* 1.03 (0.90, 1.18)* 0.77 (0.69, 0.86) 1.58 (1.40, 1.77)*

Total NNAL pmol/mgc 0.27 (0.22, 0.33) 1.31 (1.22, 1.41)* 1.14 (1.06, 1.23) * 0.84 (0.78, 0.91)* 1.03 (0.96, 1.12)*

NNN pmol/mgb 0.044 (0.31, 0.63) 0.085 (0.078, 0.093)* 0.058 (0.050, 0.064) 0.056 (0.050, 0.064) 0.093 (0.081, 0.107)*

NNN pmol/mgc 0.025 (0.017, 0.037) 0.073 (0.066, 0.081)* 0.066 (0.059, 0.072)* 0.062 (0.054, 0.070)* 0.064 (0.054, 0.076)*

1-HOP pmol/mgb 1.69 (1.23, 2.32) 1.55 (1.46, 1.65) 1.30 (1.20, 1.41) 1.53 (1.39, 1.68) 1.98 (1.77, 2.22)

1-HOP pmol/mgc 1.40 (0.99, 1.98) 1.41 (1.32, 1.49) 1.36 (1.25, 1.47) 1.56 (1.43, 1.70) 1.70 (1.52, 1.90)

Note. Data from the Population Assessment of Tobacco Use and Health (PATH) study * indicates a statistically significance difference (p < .013) from American Spirit (AS) brand a: PATH data is weighted; Parentheses include 95% confidence intervals b: Unadjusted value c: Adjusted for age, sex, race, body mass index, total nicotine equivalents (log-transformed), duration regular smoking, and time since last cigarette.

Supplementary Table 3 Median and Interquartile Ranges of Biomarkers by Data Source and Brand RCT AS Marlboro Newport Camel Pall Mall (N = 68) (N = 314) (N = 348) (N = 138) (N = 104) Total NNAL pmol/mg 0.33 (0.21, 0.63) 1.96 (1.06, 3.03) 1.06 (0.64, 1.74) 1.04 (0.56, 1.77) 1.84 (1.11, 2.57) 3-HPMA nmol/mg 5.94 (3.65, 8.20) 6.80 (4.40, 11.04) 5.95 (3.66, 8.63) 7.26 (4.68, 11.25) 8.99 (5.90, 15.55) 2-HPMA nmol/mg 0.56 (0.41, 0.94) 0.66 (0.44, 1.13) 0.57 (0.35, 0.86) 0.75 (0.50, 1.15) 0.70 (0.51, 1.07) HMPMA nmol/mg 3.48 (2.23, 5.34) 4.02 (2.68, 6.20) 3.46 (2.21, 5.07) 3.90 (2.61, 6.62) 5.50 (3.66, 7.94) CEMA nmol/mg 0.52 (0.29, 0.80) 0.70 (0.47, 1.09) 0.58 (0.38, 0.86) 0.78 (0.43, 1.20) 0.89 (0.59, 1.49) SPMA pmol/mg 3.31 (1.51, 6.49) 4.17 (1.96, 6.94) 3.17 (1.66, 5.45) 4.43 (2.40, 7.47) 4.08 (2.49, 7.96) PheT pmol/mg 2.88 (1.64, 4.45) 2.41 (1.53, 3.73) 1.74 (1.05, 2.68) 2.39 (1.53, 4.63) 2.60 (1.70, 3.66) 8-isoPGF2α pmol/mg 1.07 (0.74, 1.53) 1.21 (0.80, 1.83) 1.38 (0.91, 2.09) 1.34 (0.76, 2.10) 1.20 (0.65, 1.99) PGEM pmol/mg 45.08 (26.15, 89.47) 51.33 (29.10, 96.54) 58.78 (32.49, 117.22) 50.88 (24.96, 81.85) 48.19 (25.11, 100.52) hsCRP mg/L 1.09 (0.55, 3.83) 2.04 (0.99, 4.02) 2.25 (1.00, 5.36) 1.86 (0.77, 4.65) 2.46 (1.11, 5.13) WBC 103/µL 6.95 (5.60, 8.20) 7.70 (6.50, 9.10) 6.70 (5.50, 8.20) 7.70 (6.10, 8.90) 7.60 (6.10, 9.00) PATHa AS Marlboro Newport Camel Pall Mall (N = 392,143) (N = 9,256,656) (N = 4,428,971) (N = 1,921,109) (N = 1,970,381) Total NNAL pmol/mg 0.52 (0.19, 1.03) 1.82 (1.00, 2.81) 1.12 (0.67, 1.70) 0.84 (0.54, 1.39) 1.82 (1.21, 2.37) NNN pmol/mg 0.062 (0.025, 0.064) 0.093 (0.043, 0.161) 0.061 (0.029, 0.128) 0.063 (0.028, 0.119) 0.101 (0.053, 0.162) 1-HOP pmol/mg 1.70 (0.97, 2.09) 1.51 (1.05, 2.31) 1.32 (0.80, 2.12) 1.49 (0.96, 2.27) 1.97 (1.39, 2.68)

Note. Data from the randomized clinical trial (RCT) or the Population Assessment of Tobacco Use and Health (PATH) study a: PATH data is weighted Parentheses include interquartile range.

Tob Regul Sci.™ 2019;5(4):339-351 DOI: https://doi.org/10.18001/TRS.5.4.4 351