α,β-Unsaturated carbonyl compounds in cigarette smoke, such as acrolein and acrylamide, are considered as possible causative factors for chronic obstructive pulmonary disease (COPD) in people who smoke cigarettes. A major route of metabolism of these compounds is reaction with glutathione to form adducts that are further metabolized and excreted in the urine as mercapturic acids, which serve as useful dosimeters of exposure. In this study, we analyzed urine samples from the Singapore Chinese Health Study, a prospective epidemiology study of 63,257 men and women 45-74 years old with Chinese origin who were permanent residents or citizens of Singapore when they were enrolled in 1993-1998. A case-control study with 100 incident cases of COPD and 100 matched controls, all of whom were current cigarette smokers, was conducted within this cohort. Urine samples of the study subjects were collected during follow-up I (2000-2005) and the COPD cases were identified in follow-up II interviews (2006-2010). Urinary mercapturic acids of acrolein (3-hydroxypropyl mercapturic acid, 3-HPMA, 1), acrylamide (2-carbamoylethyl mercapturic acid, 2-CaEMA, 2), acrylonitrile (2-cyanoethyl mercapturic acid, 2-CyEMA, 3), crotonaldehyde (3-hydroxy-1-methylpropyl mercapturic acid, HMPMA-1, 4), methacrolein (3-hydroxy-2-methylpropyl mercapturic acid, HMPMA-2, 5), and methyl vinyl ketone (3-hydroxy-3-methylpropyl mercapturic acid, HMPMA-3, 6) were quantified using a LC-MS/MS method. Urinary levels of 2-CaEMA, the mercapturic acid of acrylamide, were significantly higher in cases (geometric mean 361.0 pmol/mg creatinine) than in controls (293.7 pmol/mg creatinine) and the increasing level of 2-CaEMA in tertiles was significantly associated with increased odds of developing COPD (both P <0.05), after adjustment for cigarettes per day and years of cigarette smoking. For the mercapturic acid of acrylonitrile, a weak trend of increasing 2-CyEMA with the COPD status was observed (p trend = 0.033), while none of the other mercapturic acids were significantly related to the risk of COPD. These results indicate that acrylamide in cigarette smoke should be given strong consideration as a cause of COPD in people who smoke.

Smokeless tobacco, used by more than 360 million people worldwide, is an accepted cause of cancers of the oral cavity, esophagus, and pancreas. DNA adducts are considered as critical intermediates in the carcinogenic process, but there are no published studies that have used structure-specific methods to identify and quantify oral cell DNA adducts in people who use smokeless tobacco. In the study presented here, we used liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry to identify and quantify DNA adducts in oral cells of 24 individuals who use smokeless tobacco and 25 individuals not using any tobacco or nicotine product. We quantified the following DNA adducts: (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)-one (γ-OH-Acr-dGuo) from acrolein; (6S,8S and 6R,8R)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)-one [(S,R)γ-OH-Cro-dGuo] from crotonaldehyde and/or acetaldehyde; 3-methyladenine (3-Me-Ade); 7-ethylguanine (7-Et-Gua); and 8-oxo-deoxyguanosine (8-oxo-dGuo). Levels [median (min, max)] fmol/μmol dGuo or Gua, in samples in which analytes were detected, were: γ-OH-Acr-dGuo [350 (169, 2217) vs 114 (14, 278)], (6S,8S and 6R,8R)γ-OH-Cro-dGuo [1058 (222, 2569) vs not detected], and 7-Et-Gua [2445 (1052, 5389) vs 102 (13, 627)], which were significantly greater in the oral cell DNA of individuals who use smokeless tobacco than in those not using any tobacco or nicotine product (all adjusted Ps < 0.01), while amounts of 3-Me-Ade [3739 (93, 11495) vs 1447 (318, 3596) fmol/μmol Gua, adjusted P = 0.052] and 8-oxo-dGuo [1520 (513, 5232) vs 2092 (433, 3325) pmol/μmol dGuo, adjusted P = 0.13] were not significantly different between the two groups. The sizable and significant elevations of 7-Et-Gua and (6S,8S and 6R,8R)γ-OH-Cro-dGuo in oral cell DNA when smokeless tobacco was used versus controls were particularly notable and unexpected. The results of this study provide some potentially important new data regarding DNA adduct levels in oral cells of people who use smokeless tobacco.

Previous studies have reported the existence of a direct-acting DNA ethylating agent in tobacco products; it is known that ethyl DNA adducts such as 7-ethylguanine (7-Et-Gua) are elevated in tissues of smokers, but the identity of the ethylating agent is unknown. In the study reported here, we investigated the possibility that N-ethyl-5-methyl-2-(1-methylethyl)cyclohexanecarboxamide (WS-3), a synthetic cooling agent used in tobacco and other products as a menthol alternative, could be nitrosated to produce the reactive DNA ethylating agent N-nitroso-N-ethyl-5-methyl-2-(1-methylethyl)cyclohexanecarboxamide (NO-WS-3). We synthesized NO-WS-3 and investigated its formation and stability, as well as its ability to ethylate DNA. Formation of NO-WS-3 is favored under acidic conditions that are likely to be found in the gastrointestinal tract, along with endogenous nitrite. The half-life of NO-WS-3 under biological conditions was about 16 h, ample time for its reaction with DNA to create potentially mutagenic ethyl DNA adducts which have been associated with tobacco use. Reaction of NO-WS-3 with calf-thymus DNA resulted in the formation of 7-Et-Gua (167 adducts per 108 nucleotides), 3-Et-Ade (437 adducts per 108 nucleotides), and O 6-Et-Gua (148 adducts per 108 nucleotides) well above background DNA levels in control incubations without NO-WS-3. This is the first identification of an agent potentially present in consumer products or formed in vivo that can directly ethylate DNA, providing a novel source of ethyl DNA adducts present in humans.

BACKGROUND:Banning cigarette filter ventilation has been raised by some public health researchers and organisations because it has misled people who smoke to believe they are smoking a safer cigarette. AIM:To examine the potential effects of banning ventilated filters on smoking behaviour and biomarkers of toxicant exposures. TRIAL DESIGN:Randomised controlled trial with parallel semi-blind study design. METHODS:People who smoke (n=164) ventilated cigarettes daily were randomised to smoke 'ventilated' (≈27% ventilation) or 'unventilated' (≈0.11% ventilation) study cigarettes for 6 weeks. Non-inferiority testing was conducted on the changes in urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronides (total NNAL) and cigarettes per day (CPD) between the two cigarette groups. Superiority testing was conducted for differences in smoking intensity (amount of nicotine in cigarette butts), other exposure biomarkers and subjective responses. RESULTS:The unventilated condition was generally non-inferior to the ventilated condition for change in total NNAL (3% greater for unventilated vs ventilated, less than the non-inferiority 15% margin but with wide variability with an upper bound of 95% CI of 16%, p=0.064) and in CPD (0.22 cigarettes greater for unventilated vs ventilated, lower than the non-inferiority margin of 2, p<0.001). There were no significant differences between the two groups in other biomarkers (p>0.05). Less intensity of smoking was observed with unventilated cigarettes (difference of changes=-0.04 mg nicotine, p=0.002), aligning with higher self-reported ratings of nicotine strength (p=0.005) and aversion (p=0.028). CONCLUSIONS:Overall, banning filter ventilation is unlikely to lead to greater toxicant exposures but certain subgroups might experience greater exposures.

Among cigarette smokers, higher internal smoking dose is associated with elevated lung cancer risk and mortality, independent of smoking pack-years. Some measures of epigenetic age acceleration (EAA) are associated with cigarette smoking status and exposure, as well as lung cancer risk and overall mortality. No study has examined the association between EAA measures and internal smoking dose (total nicotine equivalents (TNE; nmol/mL)), and their shared relationship with lung cancer incidence and mortality in a multiethnic population. From a subgroup of Multiethnic Cohort Study participants who smoked cigarettes at the time of biospecimen collection (n = 1969), six epigenetic clocks were computed using blood-based DNA methylation (DNAm) array data. EAA measures were computed by calculating the residuals that results from regressing an epigenetic clock on chronological age. The association of urinary TNE with EAA measures were assessed using linear regression models, adjusted for age, sex, body mass index (BMI; kg/m2), DNAm-based estimates of blood cell composition, population stratification, and self-reported pack-years of cigarette smoking. To evaluate the associations of EAA with incident lung cancer risk (n = 176 cases) and all-cause mortality (n = 780 deaths) (from 13 years of follow-up), Cox proportional hazard models, with age as the time metric, adjusted for decade of birth, sex, BMI, years of education, creatinine, DNAm-based estimates of blood cell composition, population stratification, self-reported pack-years, and urinary TNE, were used. A standard deviation (SD) increase of log-TNE was statistically significantly associated with increased AgeAccelPheno (beta = 0.416, 95

Human exposure to toxicants and carcinogens occurs via polluted air, the diet, occupational settings, endogenous processes, and tobacco products. The glutathione-S-transferase detoxification pathway resulting in the excretion of mercapturic acids is one of the most important human systems for processing and excreting toxicants and carcinogens. Maximizing effectiveness of this pathway through dietary modifications can potentially decrease the impact of such exposures. We conducted a two-site clinical trial with a crossover, single blind (to participants) design, randomized to order of product receipt comparing levels of selected urinary mercapturic acids when participants consumed a drink made from freeze-dried watercress (3× daily, 2 weeks) naturally containing 2-phenethyl isothiocyanate (PEITC) vs. a placebo drink containing maltodextrin (3× daily, 2 weeks) with a 4-week washout between the treatment periods. Two hundred forty participants (157 F) were recruited and 188 (125 F, 152 non-smokers) were compliant and completed the study. Among compliant subjects, urinary mercapturic acid detoxification products of acrolein, acrylonitrile, benzene, crotonaldehyde, methacrolein, and methyl vinyl ketone increased significantly (P ≤ 0.002), with the largest increases being observed for the mercapturic acids of acrolein (65.6%) and benzene (37.3%), both P < 0.001. Consumption of a drink, three times daily, prepared from freeze-dried watercress containing PEITC (total of approximately 40 mg/day) significantly increased the detoxification of 6 of 7 environmental toxicants and carcinogens monitored. The results indicate that watercress consumption can ameliorate exposure to environmental toxicants and carcinogens. These results are particularly timely and relevant to exposures to atmospheric pyrolysis products resulting from wildfires. Clinical trial registration number: NCT03978117.

Table S1. Urinary BOEs in PATH Study Wave 1. Table S2. Urinary BOEs of Nicotine Metabolites in PATH Study Wave 1. Table S3. Urinary BOEs of TSNAs in PATH Study Wave 1. Table S4. Urinary BOEs of PAHs in PATH Study Wave 1. Table S5. Urinary BOEs of VOCs in PATH Study Wave 1. Table S6. Urinary BOEs of Metals in PATH Study Wave 1. Table S7. Urinary BOEs of Arsenic in PATH Study Wave 1.

Obesity is a known risk factor for Pancreatic ductal adenocarcinoma (PDAC). Fatty acid binding protein 4 (FABP4) is higher in plasma of obese patients, and linked to the progression of obesity-related cancers. To provide insights into the role of FABP4 in PDAC progression and determine the potential of FABP4 inhibitor for PDAC treatment, we elucidated the anticancer mechanism of FABP4 inhibition using FABP4 null mice and FABP4 inhibitor (HTS01037). In vitro, HTS010137 suppressed FABP4-induced cell viability in mouse (KPC cells) and human PDAC cell lines. FABP4 promoted invasive potency, epithelial-mesenchymal transition (EMT), and cancer stemness markers that were associated with up-regulation of transcription factor ZEB1. In vivo, both FABP4 knockout and inhibition with HTS01037 suppressed syngeneic KPC subcutaneous tumor growth with reduction of EMT and stemness and down-regulation of ZEB1. Human xenograft growth was also inhibited by HTS01037 treatment. In an orthotopic model, HTS01037 significantly suppressed tumor growth which improved distant metastases and survivals in mice. In liver metastasis mouse model, HTS01037 attenuated development and growth of liver metastases. Moreover, HTS01037 enhanced the efficacy of gemcitabine to PDAC. These findings indicate a promising translational value of FABP4 inhibitor as a critical therapeutic option in PDAC patients.

Accumulation of the DNA adduct γ-hydroxy-1,N2-propanodeoxyguanosine (γ-OHPdG) is associated with hepatocellular carcinoma development. Theaphenon E-a green tea polyphenol extract dosed according to mg of epigallocatechin gallate-suppresses the formation of γ-OHPdG and reduces hepatocellular carcinoma development in preclinical models. This study aimed to evaluate the safety of Polyphenon E (Theaphenon E equivalent) and its effect on liver γ-OHPdG levels in patients with cirrhosis. This phase I trial used a 3 + 3 dose-escalation design with five planned Polyphenon E (epigallocatechin gallate) dose levels: 400, 800, 1,200, 1,600, and 2,000 mg daily, administered orally for 24 weeks. Each dose cohort was monitored for "discontinue therapy" criteria for 4 weeks before additional participants were enrolled in the next cohort. Participant liver samples were assessed for γ-OHPdG levels using LC/MS-MS and vibration-controlled transient elastography; endogenous catechin pharmacokinetic data were analyzed. Grade 1 and 2 treatment-related adverse events were observed in 38% of the participants. Liver γ-OHPdG levels declined after treatment in most participants. There was a decrease in the vibration-controlled transient elastography-controlled attenuation parameter in some participants. After Polyphenon E dosing, catechin pharmacokinetic clearance patterns were equivalent for all doses except 1,600 mg. Polyphenon E was well tolerated in participants with cirrhosis at a dose up to and including 1,600 mg/day. Therefore, the recommended starting dose for a phase II trial in a cirrhotic population is 1,200 mg. We observed promising Polyphenon E suppression of liver γ-OHPdG levels. PREVENTION RELEVANCE:In this phase I dose-escalation trial, Polyphenon E was well tolerated in participants with cirrhosis at a dose up to and including 1,600 mg/day. We observed promising Polyphenon E suppression of liver γ-OHPdG levels and recommend 1,200 mg dose for a future phase II trial in a cirrhotic population.

We used liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry (LC-NSI-HRMS/MS) to quantify DNA adducts released from human oral cell DNA upon neutral thermal hydrolysis followed by acid hydrolysis. The assay was applied to 80 buccal cell samples selected from those collected in the Shanghai Cohort Study, a prospective epidemiology study of 18,244 Chinese men 45-64 years old who resided in Shanghai, China when the samples were collected in 2001-2003. The DNA adducts quantified were 3-methyladenine (3-Me-Ade), 3-ethyladenine (3-Et-Ade), and 7-ethylguanine (7-Et-Gua). The method used hydrolysis of DNA samples containing the stable isotope labeled internal standards, solid phase extraction for adduct enrichment, and analysis by LC-NSI-HRMS/MS. Accuracy and precision of the analytical method were established with detection limits of 10-20 amol on column. Median levels of 3-Me-Ade -187 adducts/109 nucleotides in smokers and 129 adducts/109 nucleotides in nonsmokers; and 7-Et-Gua -49 adducts/109 nucleotides in smokers and 21 adducts/109 nucleotides in nonsmokers─were significantly higher in smokers than in nonsmokers (both P values <0.01). Levels of 3-Et-Ade -50 adducts/109 nucleotides in smokers and 43 adducts/109 nucleotides in nonsmokers - were not significantly different. These results demonstrate the applicability of a highly sensitive LC-NSI-HRMS/MS method for the analysis of human oral cell DNA for adducts released by neutral thermal and acid hydrolysis and show the significant effects of cigarette smoking on levels of 3-Me-Ade and 7-Et-Gua in this DNA. This is apparently the first study to characterize 3-Me-Ade in intact DNA isolated from any human tissue.

DNA adducts are central in the carcinogenic process because they can cause miscoding leading to permanent mutations in important genes involved in carcinogenesis. While it is known that tobacco smoking leads to increased levels of multiple DNA adducts, most DNA adducts detected to date in humans cannot be explicitly attributed to smoking but instead have various possible exogenous and endogenous sources. We plan to probe the tobacco source of DNA adducts by providing carbon-13 labelled ([13C]-labelled) cigarettes to smokers and analyzing [13C]-labelled DNA adducts in their oral cells to determine which adducts arise from smoking. Prior to conducting studies in humans, we first report here proof-of-principle machine smoking experiments to evaluate carbon isotopologues of (a) selected carbonyls and (b) DNA adducts resulting from direct exposure of cigarette smoke vapour-phase to calf-thymus DNA. The smoke of the study cigarettes, made from a 50:50 mixture of [13C]-labelled tobacco and a popular commercial tobacco, yielded similar concentrations of carbonyl compounds and their respective DNA adducts compared with the smoke of 1R6F reference cigarettes and the popular brand of cigarettes. We detected [13C]-isotopologues of DNA adducts such as 1,N6-etheno-dA, (8R/S)-3-(2’-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)-one (γ-OH-Acr-dG), and (6S,8S and 6R,8R)-3-(2’-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)-one [(6S,8S)-γ-OH-Cro-dG and (6R,8R)-γ-OH-Cro-dG], proving that they have a direct source from tobacco smoke and providing important new insights regarding their mechanisms of formation. These unique results form the basis for further studies in cell culture and in cigarette smokers to establish how carcinogens in tobacco smoke cause DNA adduct formation.

A method was developed for the quantitation of propylene glycol in human saliva as a potentially useful biomarker for e-cigarette use. Saliva was collected from e-cigarette users, cigarette smokers, and non-users of any tobacco or nicotine product with approval from the University of Minnesota Human Research Protection Program Institutional Review Board. Two separate protocols, termed programmed and non-programmed, were used. In the programmed protocol, e-cigarette users (n = 20) and cigarette smokers (n = 20) brushed their teeth, waited 15 min, then used their product every 30 s for 5 min and collected a 2 mL saliva sample immediately after the last puff. In the non-programmed protocol, cigarette smokers (n = 30) and e-cigarette users (n = 21) came to the clinic and directly provided a saliva sample for analysis. Non-users (n = 29) of any nicotine or tobacco product served as controls. Propylene glycol in saliva was quantified by gas chromatography-mass spectrometry as its heptafluorobutyrate ester, using propylene glycol-d8 as the internal standard. Accuracy and precision of the assay were established. Levels of salivary propylene glycol (mean ± S.D.) were 269 ± 319 and 8.04 ± 8.22 μg/mL saliva in e-cigarette users and cigarette smokers, respectively, in the programmed protocol, while the corresponding levels in the non-programmed protocol were 4.94 ± 8.68 and 1.91 ± 1.99 μg/mL. These amounts were all significantly greater than those found in non-users of any tobacco or nicotine product (0.39 ± 0.32 μg/mL). This straightforward validated assay is expected to be useful in future clinical studies of e-cigarette use.

[This corrects the article DOI: 10.1016/j.lana.2024.100796.].

Supplemental Table 3. Pearson Correlation Coefficients (R) for smoking-related urinary biomarkers

Supplemental Table 7. Association of urinary biomarkers and lung cancer risk in Southern Community Cohort participants stratified by histologic cell-type

Supplemental Table 1. Coefficients of Variation for Biomarker analysis

Exposure to polycyclic aromatic hydrocarbons (PAHs), byproducts of incomplete combustion, and their effects on the development of cancer are still being evaluated. Recent studies have analyzed the relationship between PAHs and tobacco or dietary intake in the form of processed foods and smoked/well-done meats. This study aims to assess the association of a blood biomarker and metabolite of PAHs, r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT), dietary intake, selected metabolism SNPs, and pancreatic cancer. Demographics, food-frequency data, SNPs, treatment history, and levels of PheT in plasma were determined from 400 participants (202 cases and 198 controls) and evaluated based on pancreatic adenocarcinoma diagnosis. Demographic and dietary variables were selected based on previously published literature indicating association with pancreatic cancer. A multiple regression model combined the significant demographic and food items with SNPs. Final multivariate logistic regression significant factors (p-value < 0.05) associated with pancreatic cancer included: Type 2 Diabetes [OR = 6.26 (95% CI = 2.83, 14.46)], PheT [1.03 (1.02, 1.05)], very well-done red meat [0.90 (0.83, 0.96)], fruit/vegetable servings [1.35 (1.06, 1.73)], recessive (rs12203582) [4.11 (1.77, 9.91)], recessive (rs56679) [0.2 (0.06, 0.85)], overdominant (rs3784605) [3.14 (1.69, 6.01)], and overdominant (rs721430) [0.39 (0.19, 0.76)]. Of note, by design, the level of smoking did not differ between our cases and controls. This study does not provide strong evidence that PheT is a biomarker of pancreatic cancer susceptibility independent of dietary intake and select metabolism SNPs among a nonsmoking population.

The U.S. Food and Drug Administration has stated its intention to reduce the nicotine content of combustible cigarettes to render them less addictive. This study evaluated the impact of providing adults who smoke with both very low nicotine content cigarettes (VLNCCs) and electronic cigarettes (ECs) of varying nicotine content on measures of human abuse potential. Participants (n = 213) were adult combustible cigarette users. They smoked their usual brand cigarettes (UBCs) during Phase 1 (baseline; week 1) and were provided with and encouraged to exclusively use VLNCCs during Phase 2 (weeks 2-4). During dual-product Phases 3 (weeks 5-7) and 4 (weeks 8-10), participants received both VLNCCs and ECs (assigned to one of two EC devices in higher or lower nicotine concentrations and choice of flavor), with instructions to use them freely in Phases 3 and 4. Assessments included product use, exposure, acceptability, risk perception, and withdrawal-related measures. Results indicated that participants used significantly fewer UBCs during the VLNCC and dual-product phases and smoked fewer VLNCCs during the dual-product phases than the VLNCC-only phase. Neither EC liquid nicotine concentration nor flavor influenced product use. The three study product phases resulted in less product liking and more withdrawal symptoms than the UBC phase. These results suggest that adults who smoke are able to switch much of their tobacco product use from UBCs to VLNCCs and will substitute combustible UBCs and VLNCCs with noncombustible nicotine-containing ECs, but most remain dual users, at least in the short term.

Supplemental Table 2. Median and SD of the log distribution for smoking-related urinary biomarkers overall