Research Cigarettes Research Articles

Racial/ethnic differences in the acute effects of reduced nicotine content cigarettes among adolescents who smoke

ObjectiveReducing the nicotine content in cigarettes decreases their addictiveness and abuse liability, including among adolescents. Whether these effects differ by race/ethnicity is unknown. This study is a secondary analysis of previously published data collected between 2014–2017. We examined racial/ethnic differences in the effects of smoking cigarettes with varying nicotine content levels on subjective effects and tobacco withdrawal among adolescents who smoke daily. MethodsAcross two counterbalanced sessions, 50 adolescents recruited from Rhode Island (ages 15–19; 30 % Underrepresented Minorities [URM], 20 % Asians and Pacific Islanders [API]; 50 % Non-Hispanic Whites) self-administered a very low nicotine content (VLNC; 0.4 mg nicotine/g of tobacco) or normal nicotine content control (NNC; 15.8 mg/g) research cigarette following overnight abstinence. Subjective effects were reported post-administration and tobacco withdrawal outcomes were calculated from pre- to post-administration scores. Multilevel linear models tested main and interactive effects between cigarette nicotine content and race/ethnicity on all study outcomes. ResultsParticipants reported lower positive subjective effects and reductions in smoking urges after smoking a VLNC cigarette relative to smoking an NNC cigarette (ps < 0.01). A main effect of race/ethnicity emerged, such that API (vs. URM and White) adolescents reported lower positive subjective effects, greater craving reduction, and higher cigarette aversion after smoking, regardless of nicotine content (ps < 0.05). Significant interactions were found between race/ethnicity and nicotine content for cigarette aversion, such that API adolescents rated VLNC (vs. NNC) cigarettes as less aversive than White and URM adolescents did (p = 0.03). ConclusionsFindings provide evidence that VLNC cigarettes may reduce abuse liability and tobacco withdrawal symptoms for adolescents across racial/ethnic groups and particularly for API youth.

Free Radicals in Little Cigar Mainstream Smoke and the Potential Influence of Flavoring Chemicals on Free Radical Production.

Implementation of the Tobacco Control Act in 2009 banned characterizing flavors in cigarettes (except menthol and tobacco), but substitution has occurred by the continued availability of alternative flavored products (i.e., flavored little cigars). Little is known about how flavorants in noncigarette tobacco products impact human health. Thus, we investigated the impact of flavorants on free radical production in the mainstream smoke of little cigars. Gas- and particulate-phase free radical yields in mainstream smoke generated from 12 commercial little cigar brands and research little cigars and cigarettes were measured via electron paramagnetic resonance spectroscopy using the International Organization of Standardization (ISO) smoking protocol. Flavorants were extracted from unsmoked little cigars and analyzed by gas chromatography-mass spectroscopy. Gas- and particulate-phase radical yields from little cigars ranged from 13.5 to 97.6 and 0.453-1.175 nmol/unit, respectively. Comparatively, research cigarettes yielded an average of 4.9 nmol gas-phase radicals/unit and 0.292 nmol particulate-phase radicals/unit. From the products, 66 flavorants were identified, with each brand containing 4-24 individual flavorants. The free radical content was strongly correlated with the number of flavorants present in each cigar (r = 0.74, p = 0.01), indicating that highly flavored little cigars may produce higher levels of toxic free radicals. The presence of the flavorant ethyl methylphenylglycidate (strawberry) was associated with >2-fold higher levels of GP radicals (p = 0.001). Our results show that free radical delivery from little cigars is greater than that from research cigarettes and provide empirical evidence for the harmfulness of flavored tobacco products. Additionally, it demonstrates that flavorants present in combustible tobacco products can influence the levels of free radicals produced. Therefore, future tobacco product standards should consider little cigars.

Reactions to a Nicotine Reduction Policy Among Adolescents Who Smoke: A Qualitative Study.

The Biden administration is pursuing a nicotine reduction policy in the U.S. to render cigarettes less addictive. In this study, we qualitatively investigated adolescents' subjective responses to very low nicotine content (VLNC) cigarettes, reasons for incomplete adherence to using them, and their expected responses to a nicotine reduction policy. Adolescents who smoke cigarettes daily (ages 15-19; N=60) were enrolled in a three-week double-blind randomized clinical trial and assigned to smoke either normal nicotine content (NNC) or VLNC research cigarettes. Following the trial, 52 participants completed qualitative interviews about their reactions to the cigarettes and to the idea of a nicotine reduction policy. We utilized a template style approach to thematic analysis involving immersion in the data, codebook development with both inductive and deductive codes, and iterative refinement of themes. Reasons for incomplete adherence to smoking only research cigarettes focused on dislike for cigarette taste and lower satisfaction compared to usual brand cigarettes. Negative evaluations of research cigarettes were common across both groups. Many participants in both groups reported that they would decrease their smoking or quit entirely if the research cigarettes were the only ones legally available for purchase in the U.S. Adolescents may respond to a cigarette nicotine reduction policy by decreasing their cigarette smoking and eventually quitting. These findings suggest a need for public health strategies to reduce smoking initiation and progression in young people and to encourage cessation in the context of a nicotine reduction policy. Participants' negative subjective responses and challenges with research cigarette adherence offer insight into factors that might influence young people's reactions to a real-world nicotine reduction policy. Adolescents who smoke may increase their use of alternative tobacco products, especially e-cigarettes, if this policy were implemented. Specifically, themes identified across participant responses highlight important considerations for how such a policy might be implemented with specific attention to the unique smoking behaviors and needs of this vulnerable population.

Nicotinic Receptor Alpha-5 Subunit Gene Polymorphism is Associated With Heavy Smoking Under a Range of Nicotine Dosing Conditions.

This study aimed to assess the role of the rs16969968 variant of nicotinic receptor alpha-5 subunit in regulating smoking behavior and nicotine intake in response to nicotine manipulations among dependent smokers in a naturalistic environment. Sixty-nine adults (19 females) smoking 10 or more cigarettes per day (CPD) were asked to complete four 2-week study phases during which they smoked exclusively one of two types of Spectrum nicotine research cigarettes (FTC nicotine yield 0.8 and 1.6 mg, respectively), their usual brand of cigarettes, or their usual brand of cigarettes while wearing a 21-mg nicotine patch. Measurements included rs16969968 genotype, number of CPD, smoking topography, and plasma cotinine. Compared to controls (G/G carriers), A allele carriers reported smoking 4 to 5 more CPD across all conditions (all ps < .05). Mean total smoke volume per day and cotinine were greater in A allele carriers than in controls (ps = .05, .046, respectively). No significant genotype differences were found in smoking compensation indices for the switch from medium to high-nicotine-yield cigarettes. Nicotine patch-induced reductions in cigarettes smoked per day and total smoke volume per day showed significant interactions between genotype and pre-patch levels, with heavier smokers showing greater effects of genotype (p = .052 and p = .006, respectively). Results suggest that the rs16969968 variants regulate the heaviness of smoking primarily by their impact on daily numbers of cigarettes smoked, but no genotype differences were found in smoking compensation after switching from medium to high-nicotine cigarettes. The differences in daily cigarette consumption between rs16969968 risk-allele carriers and controls are shown to be consistent regardless of manipulations of cigarette nicotine content and transdermal nicotine supplementation and markedly greater among dependent smokers than those observed in the general smoker populations. G/G allele carriers, relative to A allele carriers, appeared to be more sensitive to the nicotine patch manipulation, reducing their smoking to a greater extent. These findings support continued efforts in the development of personalized intervention strategies to reduce the rs16969968-conveyed genetic propensity for heavy smoking.

Role of reactive carbonyls and superoxide radicals in protein damage by cigarette smoke extracts: Comparison of Heat-not-Burn e-cigarettes to conventional cigarettes

Oxidative protein damage involving carbonylation of respiratory tract proteins typically accompanies exposure to tobacco smoke. Such damage can arise via multiple mechanisms, including direct amino acid oxidation by reactive oxygen species or protein adduction by electrophilic aldehydes. This study investigated the relative importance of these pathways during exposure of a model protein to fresh cigarette emission extracts. Briefly, protein carbonyl adducts were estimated in bovine serum albumin following incubation in buffered solutions with whole cigarette emissions extracts prepared from either a single 1R6F research cigarette or a single “Heat-not-Burn” e-cigarette. Although both extracts caused concentration-dependent protein carbonylation, conventional cigarette extracts produced higher adduct yields than e-cigarette extracts. Superoxide radical generation by conventional and e-cigarette emissions was assessed by monitoring nitro blue tetrazolium reduction and was considerably lower in extracts made from “Heat-Not-Burn” e-cigarettes. The superoxide dismutase/catalase mimic EUK-134 strongly suppressed radical production by whole smoke extracts from conventional cigarettes, however, it did not diminish protein carbonyl adduction when incubating smoke extracts with the model protein. In contrast, edaravone, a neuroprotective drug with strong carbonyl-trapping properties, strongly suppressed protein damage without inhibiting superoxide formation. Although these findings require extension to appropriate cell-based and in vivo systems, they suggest reactive aldehydes in tobacco smoke make greater contributions to oxidative protein damage than smoke phase radicals.

Abstract 4830: Predicting drug treatment response in African American lung cancer patients who smoke

Abstract Background: Lung cancer is the third most commonly diagnosed cancer in the US and the leading cause of cancer-related death. African Americans (AAs) have higher lung cancer incidence and mortality rates, and lower survival rates, when compared to their European American (EA) counterparts. It is widely known that cigarette smoking is the strongest risk factor for lung cancer development, but it also plays a significant role in clinical outcome. Cigarette smoking after lung cancer diagnosis (up to 30% patients) comes with several risks, including increased secondary tumors and mortality rates (by 20%), as well as poor treatment outcomes (higher radiation and chemotherapy failure and higher drug clearance). There are limited transcriptomic studies that describe molecular characterizations of AA smokers with lung cancer to predict treatment outcomes. Hypothesis: Cigarette smoking leads to greater drug resistance-related gene expression in AA lung cancer patients. Methods: mRNA expression changes in an AA (NCI-H2172) and EA (NCI-H1944) lung cancer cell line pair exposed to liquid cigarette smoke condensate (CSC) were profiled. The cell lines were matched based on age (62 years), sex (female), histology (lung adenocarcinoma, LUAD, the most common subtype), and culture properties (adherent). The CSC was derived from Murty Pharmaceuticals and prepared by smoking University of Kentucky's 3R4F Standard Research Cigarette on an FTC Smoke Machine. A total of 500,000 cells per well were treated for one hour under standard incubation conditions (37°C, pH 7.0, 5% CO2). Cells were pelleted, RNAs were isolated, treated with DNAse-I to remove contaminating genomic DNA, quantified, and sent for bulk mRNA-sequencing (Novogene Co). Three biological replicates and technical triplicates were performed. Partek Flow and Partek Genomics Suite workflows were used to identify population-specific differentially expressed gene (DEG) signatures and pathway enrichment after CSC exposure. DEG were profiled in LUAD tissues from current smokers in an AA and EA TCGA cohort (n = 18 AAs, 86 EAs). Results: Population-specific gene expression changes were discovered (n = 52 AA DEGs, n = 31 EA DEGs; P &amp;lt;0.001). All of the DEGs were mutually exclusive with no overlap. Some pathways were shared between both populations and others were unique. PI3K/Akt/mTOR signaling pathway enrichment was unique in AA LUAD cells and ECM receptor interactions were distinct in EA LUAD cells. AKT2 (which activates mTOR) had significantly higher expression in LUAD tumor tissues from current AA smokers compared to current EA smokers. Conclusion: Population-specific PI3K/Akt/mTOR pathway alterations may help explain drug resistance in LUAD tumors from AA patients. PI3K signaling is considered a significant cause of resistance to chemotherapy, targeted therapy, and immunotherapy. Future Directions: Expose AA and EA LUAD cell lines to CSC and PI3K/Akt/mTOR pathway inhibitors. Citation Format: Mia Ray, Isaiah Osei-Gyening, Kwabena Acheampong, Marisa Riebesell, Khadijah A. Mitchell. Predicting drug treatment response in African American lung cancer patients who smoke [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4830.

Dermal thirdhand smoke exposure induced epidermal alterations in human keratinocyte cells through oxidative damage and MMP-1 expression.

Thirdhand smoke (THS) is the residual cigarette smoke that settles on indoor surface fabrics, dust and can accumulate in the environment. Therefore, it can be a risk factor for individuals who have frequent dermal contact with THS-contaminated surfaces. In the present study, it was aimed to elucidate the toxicity of dermal THS exposure in HaCaT human keratinocytes. The THS was extracted from terrycloth exposed to 3R4F research cigarette smoke in a closed chamber and the adverse outcomes induced by THS were determined through assessment of cytotoxicity tests (MTT and NRU), intracellular GSH level, total SOD activity, matrix metalloproteinase-1 (MMP-1) and IL-6 levels. The wound healing capacity of THS-exposed keratinocytes was evaluated via scratch assay. A potent antioxidant isothiocyanate compound, sulforaphane (SFN), was used as a negative control. THS was dose-dependently cytotoxic (12.5%-100%, v/v) to the HaCaT cells through mitochondrial cell dysfunction (p < 0.01), which was ameliorated by SFN (0.62 μM) pre-treatment. In parallel, THS exposure significantly decreased the intracellular GSH deposits and T-SOD activity in keratinocytes. Collagen degradation through elevated MMP-1 expression was observed in THS-exposed cells in parallel with the delay of wound healing and increased pro-inflammatory response in a dose-dependent manner (p < 0.05). The findings are expected to raise awareness about THS as an environmental pollutant for skin, particularly in the highest-ranked countries in cigarette consumption. To conclude, these results might contribute to the studies on the importance of dermal exposure to THS in the pathogenesis of epidermal alterations and the other skin diseases.

Abstract 649: Comparative effects of combustible cigarette versus electronic cigarette exposures on K-ras mutant lung cancer

Abstract Combustible cigarette smoking (CCS) is causally related to ~90% of all lung cancers and induces various tumor-initiating effects, some of which (e.g., inflammation) are not reversible even after smoking cessation. More recently, use of non-combustible smoking vectors, such as electronic cigarette vapors (ECV), have rapidly increased, especially among youth. Despite this emerging public health concern, the long-term impact of ECV exposure is poorly understood. Our lab sought to assess the safety of ECV and determine their influence on immune dysfunction and lung cancer progression by comparing the effects of CCS and ECV in an airway epithelial specific K-ras mutant mouse model of lung adenocarcinoma (CC-LR). Three cohorts of 6-week-old CC-LR mice were exposed to either room air, CCS, or ECV 5 days per week for 8 weeks. For CCS exposure, mice were exposed to CCS from 3R4F research cigarettes at a rate of 3 puffs/min for 2 hours each day using the SCIREQ integrated Cigarette Smoking Robot. For ECV exposure, mice were exposed to 72 mg/ml of liquid nicotine in 50% propylene glycol/vegetable glycerin solution at a rate of 3 puffs/min for 2 hours using the SCIREQ Electronic Nicotine Delivery System. While CCS led to a significant increase in tumor burden (25%), ECV showed no significant changes compared to room air-exposed control. Bronchoalveolar lavage fluid had significantly increased total lung immune cell infiltrates in both CCS (30%) and ECV (25%), particularly in macrophage and lymphocyte populations. Immunophenotyping of CCS and ECV exposed lungs displayed pronounced pro-tumor immunosuppressive phenotypes, characterized by significantly decreased CD4+ IFNγ+ and CD8+ GZMB+ cytotoxic T cells along with a significantly elevated CD4+ FOXP3+ regulatory T cells. Furthermore, the cytotoxic cytokine IFNγ was significantly reduced in lungs of CCS- (7-fold) and ECV-exposed (2-fold) mice compared to room air-exposed controls. We also found increased levels of the immunosuppressive cytokine IL-10 in lungs of CCS- and ECV-exposed mice. Previous studies have shown IL-10 to play an important role in microbiome-associated immune modulation, hence we performed a preliminary gut and lung microbiome study to assess microbiome dissimilarity in these cohorts. Taxonomic profiling via 16S rRNA gene sequencing of matched stool and lung samples showed differences in the relative abundance of several lung Proteobacteria spp. while gut Firmicutes, particularly Turicibacter and Ileibacterium, were increased by CCS and ECV. In conclusion, while both CCS and ECV increased immune suppression, ECV did not significantly promote tumorigenesis during this timeframe of intervention. Future studies probing differences in microbiome-modulated CCS and ECV immune dysfunction can help pave way for identification of new targets that foster new interception and early treatment strategies for K-ras mutant lung cancer. Citation Format: Walter V. Velasco Torrez, Maria T. Grimaldo, Michael J. Clowers, Bo Yuan, Segundo del Aguila Soto, Iman Bouchelkia, Javier Eduardo Moreno Barragan, Umesh C. Karandikar, Joseph F. Petrosino, Florencia McAllister, Humam Kadara, Kristi Louise Hoffman, Seyed Javad Moghaddam. Comparative effects of combustible cigarette versus electronic cigarette exposures on K-ras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 649.

A contextualised e-cigarette testing strategy shows flavourings do not impact lung toxicity in vitro

Vaping has the potential to reduce the individual health risks associated with smoking and e-cigarette flavours have been reported to help smokers’ transition from cigarettes. In this manuscript, we provide evidence to support the reduced risk potential of e-cigarette aerosols and flavours by assessing commercially available e-liquids (Vuse ePod – Manufactured by British American Tobacco) in a 2D in vitro screening approach. We also analysed selected flavours using a more physiologically relevant 3D (MucilAir) whole aerosol exposure model, measuring toxicity and functional endpoints such as Trans Epithelial Electrical Resistance, Cilia Beat Frequency and Active Area. To contextualise responses, we have compared e-cigarette aerosol to cigarette smoke (1R6F research cigarette) and calculated the percentage reduction using a point of departure approach. We show that aerosolised flavoured e-liquids, (appropriately stewarded) do not increase the overall measured aerosol toxicity when compared to cigarette smoke. In fact, we demonstrate that the measured in vitro cellular toxicity of flavoured e-cigarette products remains > 95% reduced when compared to cigarette smoke toxicity, using point of departure (IC80) approach. These data indicate that the overall product toxicity is not increased in a flavour dependent manner and that flavoured e-cigarette products can potentially play a role in tobacco harm reduction.

Qualitative reactions to a low nicotine product standard for cigarettes from adolescents and young adults living in the United States who smoke

The Biden Administration is considering a low nicotine product standard for cigarettes. This qualitative study examined reactions to a nicotine reduction policy among adolescents and young adults (AYA) who smoke cigarettes. After completing a lab study involving masked exposure either to low nicotine or normal nicotine research cigarettes and unmasked exposure to e-cigarettes varying in nicotine concentration and flavor, we conducted follow-up semi-structured interviews (N = 25) to explore participants’ knowledge, attitudes, and perceptions of a low nicotine product standard and their anticipated tobacco use behavior after policy implementation. Interviews were audio-recorded, transcribed verbatim, double-coded, and analyzed using reflexive thematic analysis. Nearly half of participants supported the policy because they thought it would prevent young people from starting smoking and/or would help people quit. Reasons some participants opposed the policy included beliefs that adults should have the choice to smoke or that a nicotine reduction policy is counterintuitive because the government benefits from cigarette sales. Others believed the policy would be ineffective because youth could circumvent the policy (e.g., illicit market) or would increase their smoking to maintain the same nicotine level. Almost half of participants said they would quit smoking while the other half said they would continue smoking, although potentially reduce their smoking. Overall, our qualitative findings point to the need for pre-policy media campaigns targeting AYA who smoke to minimize negative reactions, dispel fears, and correct misperceptions as well as encourage quitting and provide information on accessing cessation resources.

A Method to Amend Cigarettes With Menthol for Use in Research.

Studies have evaluated the role of menthol cigarettes on various addiction-related outcomes; however, the effect of varying menthol content on these outcomes has not been evaluated. We developed a method to amend non-menthol SPECTRUM Research Cigarettes to contain menthol at four different levels. SPECTRUM Research Cigarettes, NRC 600 (0.8 mg nicotine; 10 mg tar), were modified to contain target menthol amounts at 3, 6, and 12 mg/cigarette by injecting 25 µL ethanol/triacetin/menthol solutions of varying concentrations (120 mg menthol/mL, 240 mg/mL, and 480 mg/mL) into four distinct locations in the filter and tobacco rod. Menthol content was tested in triplicate in the whole cigarette and in the tobacco rod and filter at 1, 24, 48, and 72 hours for each target menthol level using an extraction solution of quinoline in methyl-tert-butyl ether and measured using gas chromatography with flame ionization detection. Injections into the filter and tobacco rod (12.5 µL each) yielded equal menthol distribution up to 72 hours. However, total menthol content decreased from an average of 90.3% of the target menthol concentration at 1 hour to 80.7% at 72 hours in cigarettes stored individually in glass tubes at room temperature. Analysis of urinary menthol glucuronide confirmed that amended cigarettes used within 24 hours of injection delivered dose-related menthol levels to participants in a clinical laboratory setting. This method can be used to modify cigarettes with a range of reliable menthol levels in both filter and tobacco rod for use in laboratory and clinical research. This study presents a technique for modifying cigarettes with different levels of menthol that can reliably deliver dose-related menthol levels to participants when smoked in a clinical study. The technique can be used to quickly amend cigarettes to examine the independent effects of varying flavor and additive levels on smoking behavior, nicotine pharmacokinetics, mainstream smoke emissions, and other laboratory or clinical research outcomes.

Conventional tobacco products harbor unique and heterogenous microbiomes

While an increasing number of studies have evaluated tobacco microbiomes, comparative microbiome analyses across diverse tobacco products are non-existent. Moreover, to our knowledge, no previous studies have characterized the metabolically-active (live) fraction of tobacco bacterial communities and compared them across products. To address these knowledge gaps, we compared bacterial communities across four commercial products (cigarettes, little cigars, cigarillos and hookah) and one research cigarette product. After total DNA extraction (n = 414) from all samples, the V3V4 region of the 16S rRNA gene was sequenced on the Illumina HiSeq platform. To identify metabolically-active bacterial communities within these products, we applied a coupled 5-bromo-2′-deoxyuridine labeling and sequencing approach to a subset of samples (n = 56). Each tobacco product was characterized by its signature microbiome, along with a shared microbiome across all tobacco products consisting of Pseudomonas aeruginosa, P. putida, P. alcaligenes, Bacillus subtilis, and Klebsiella pneumoniae. Comparing across products (using Linear discriminant analysis Effect Size (LEfSe)), a significantly higher (p < 0.05) relative abundance of Klebsiella and Acinetobacter was observed in commercial cigarettes, while a higher relative abundance of Pseudomonas and Pantoea was observed in research cigarettes. Methylorubrum and Paenibacillus were higher in hookah, and Brevibacillus, Lactobacillus, Bacillus, Lysinibacillus, and Staphylococcus were higher in little cigars and cigarillos. Across all products, the majority of the metabolically-active bacterial communities belonged to the genus Pseudomonas, followed by several genera within the Firmicutes phylum (Bacillus, Terribacillus, and Oceanobacillus). Identification of some metabolically-active pathogens such as Bacillus cereus and Haemophilus parainfluenzae in commercial products is of concern because of the potential for these microorganisms to be transferred to users' respiratory tracts via mainstream smoke. Future work is warranted to evaluate the potential impact of these tobacco bacterial communities on users' oral and lung microbiomes, which play such an important role on the spectrum from health to disease.

Impairment of Endothelial Function by Cigarette Smoke Is Not Caused by a Specific Smoke Constituent, but by Vagal Input From the Airway.

Exposure to tobacco or marijuana smoke, or e-cigarette aerosols, causes vascular endothelial dysfunction in humans and rats. We aimed to determine what constituent, or class of constituents, of smoke is responsible for endothelial functional impairment. We investigated several smoke constituents that we hypothesized to mediate this effect by exposing rats and measuring arterial flow-mediated dilation (FMD) pre- and post-exposure. We measured FMD before and after inhalation of sidestream smoke from research cigarettes containing normal and reduced nicotine level with and without menthol, as well as 2 of the main aldehyde gases found in both smoke and e-cigarette aerosol (acrolein and acetaldehyde), and inert carbon nanoparticles. FMD was reduced by all 4 kinds of research cigarettes, with extent of reduction ranging from 20% to 46% depending on the cigarette type. While nicotine was not required for the impairment, higher nicotine levels in smoke were associated with a greater percent reduction of FMD (41.1±4.5% reduction versus 19.2±9.5%; P=0.047). Lower menthol levels were also associated with a greater percent reduction of FMD (18.5±9.8% versus 40.5±4.8%; P=0.048). Inhalation of acrolein or acetaldehyde gases at smoke-relevant concentrations impaired FMD by roughly 50% (P=0.001). However, inhalation of inert carbon nanoparticles at smoke-relevant concentrations with no gas phase also impaired FMD by a comparable amount (P<0.001). Bilateral cervical vagotomy blocked the impairment of FMD by tobacco smoke. There is no single constituent or class of constituents responsible for acute impairment of endothelial function by smoke; rather, we propose that acute endothelial dysfunction by disparate inhaled products is caused by vagus nerve signaling initiated by airway irritation.

Tobacco smoke exacerbates Filifactor alocis pathogenicity.

Filifactor alocis has recently emerged as a periodontal pathobiont that appears to thrive in the oral cavity of smokers. We hypothesized that identification of smoke-responsive F. alocis genes would provide insight into adaptive strategies and that cigarette smoke would enhance F. alocis pathogenesis in vivo. F. alocis was grown in vitro and cigarette smoke extract-responsive genes determined by RNAseq. Mice were exposed, or not, to mainstream 1R6F research cigarette smoke and infected with F. alocis, or not, in an acute ligature model of periodontitis. Key clinical, infectious, and immune data were collected. In culture, F. alocis growth was unaffected by smoke conditioning and only a small number of genes were specifically regulated by smoke exposure. Reduced murine mass, differences in F. alocis-cognizant antibody production, and altered immune profiles as well as altered alveolar bone loss were all attributable to smoke exposure and/or F. alocis infection in vivo. F. alocis is well-adapted to tobacco-rich conditions and its pathogenesis is enhanced by tobacco smoke exposure. A smoke-exposed ligature model of periodontitis shows promise as a tool with which to further unravel mechanisms underlying tobacco-enhanced, bacteria-induced disease.

Leveraging the cigarette purchase task to understand relationships between cumulative vulnerabilities, the relative reinforcing effects of smoking, and response to reduced nicotine content cigarettes

We examined if the relative-reinforcing effects of smoking increase with greater cumulative vulnerability and whether cumulative vulnerability moderates response to reduced nicotine content cigarettes. Participants were 775 adults from randomized clinical trials evaluating research cigarettes differing in nicotine content (0.4, 2.4, 15.8 mg/g). Participants were categorized as having low (0–1), moderate (2–3), or high (≥4) cumulative vulnerability. Vulnerabilities included rural residence, opioid use disorder, affective disorder, low educational attainment, poverty, unemployment, and physical disability. We used the cigarette purchase task (CPT) to assess the relative-reinforcing effects of participants' usual-brand cigarettes at baseline and study cigarettes during the 12-week trial. The CPT is a behavioral-economic task wherein participants estimate likely smoking (demand) over 24 h under escalating cigarette price. Demand is characterized by two factors: Amplitude (demand volume at zero/minimal price) and Persistence (demand sensitivity to price). Greater cumulative vulnerability was associated with greater demand Amplitude (F[2709] = 16.04,p < .0001) and Persistence (F[2709] = 8.35,p = .0003) for usual-brand cigarettes. Demand Amplitude for study cigarettes increased with increasing cumulative vulnerability (F[2619] = 19.59, p < .001) and decreased with decreasing nicotine content ([4879] = 5.45, p < .001). The only evidence of moderation was on demand Persistence (F[8867] = 2.00,p = .04), with larger reductions at the 0.4 mg/g compared to 15.8 mg/g doses among participants with low compared to moderate or high cumulative vulnerability. The relative-reinforcing effects of smoking clearly increase with greater cumulative vulnerability. Reducing nicotine content would likely reduce demand Amplitude across cumulative-vulnerability levels but reductions in demand Persistence may be more limited among those with greater cumulative vulnerability.

Smoking abstinence and cessation-related outcomes one month after an immediate versus gradual reduction in nicotine content of cigarettes

The United States Food and Drug Administration has the authority to reduce the nicotine content in cigarettes to minimal or non-addictive levels and could do so immediately or gradually over time. A large clinical trial compared the two approaches. This secondary analysis assesses abstinence and cessation-related outcomes one month after the trial concluded, when participants no longer had access to very low nicotine content (VLNC) research cigarettes. Smokers not interested in quitting (N = 1250) were recruited for the parent trial from 2014 to 2016 across 10 sites throughout the US and randomized to a 20-week study period during which they immediately switched to VLNC cigarettes, gradually transitioned to VLNC cigarettes with five monthly dose reductions, or smoked normal nicotine research cigarettes (control). At the one-month follow-up, both immediate and gradual reduction resulted in greater mean cigarette-free days (4.7 and 4.6 respectively) than the control group (3.2, both p < .05). Immediate reduction resulted in fewer mean cigarettes per day (CPD = 10.3) and lower Fagerström Test for Cigarette Dependence (FTCD = 3.7) than the gradual (CPD = 11.7, p = .001; FTCD = 3.8, p = .039) and control (CPD = 13.5, p < .001; FTCD = 4.0, p < .001) groups. Compared to controls, gradual reduction resulted in reduced CPD (p = .012) but not FTCD (p = .13). Differences in CO-verified 7-day point-prevalence abstinence were not significant. Findings demonstrate that switching to VLNC cigarettes resulted in reduced smoking and nicotine dependence severity that was sustained for at least a month after the VLNC trial period in smokers who were not interested in cessation. The greatest harm reduction endpoints were observed in those who immediately transitioned to VLNC cigarettes.

A comparison of nicotine content methods to produce a UPC2-MS2 method for the analysis of nicotine and minor alkaloids in SPECTRUM nicotine research cigarettes.

Nicotine is the principal alkaloid in tobacco and has been the primary subject of scientific investigation for its pharmacological effects contributing to tobacco use, dependence, withdrawal, and physical harm. Related minor alkaloids, accounting for less than 6% of alkaloid content in tobacco leaves, may also mirror some of the same pharmacological effects. To detect such low concentrations of the minor alkaloids, tobacco product methods produced by the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) using gas chromatography and flame ionization detection (GC-FID) have been adapted for use with gas chromatography-mass spectrometry (GC-MS). Nicotine and minor alkaloid content in SPECTRUM Nicotine Research Cigarettes (NRC) have previously been determined using GC-FID; however, the minor alkaloids were unable to be detected or quantitated. This study employed UltraPerformance Convergence Chromatography (UPC2) system coupled with tandem mass spectrometry (MS2) to determine the nicotine and minor alkaloid content in NRC tobacco products. CORESTA Recommended Methods (CRMs) were adapted for their sample preparative procedures for optimal extraction followed by detection with UPC2-MS2. These results were compared to two separate CRMs that used GC-FID and GC-MS2 as well as an alternative method with GC-MS2 detection. The GC-FID and GC-MS2 CRM preparations along with the alternative GC-MS2 were unable to detect the analytes in every NRC formulation, whereas the UPC2-MS2 extraction and detection method was able to quantify every analyte in every NRC formulation. This increased sensitivity demonstrates the utility of the UPC2-MS2 analytical method in accurately detecting and quantifying nicotine and minor alkaloids in tobacco filler.

Pro‐Inflammatory Profiling Of RAGE Targeted Mice Following Short‐Term Tobacco Exposure

Inflammation underpins pulmonary disease progression during tobacco smoke exposure that may culminate in irreversdible pulmonary remodeling. While primary smoke poses notable risk, nearly half of the US population is also at risk due to exposure to secondhand smoke (SHS). In the present study, we assessed a potential role for RAGE, a cell‐surface pattern recognition receptor implicated in pro‐inflammatory signaling, following exposure to SHS. Specifically, we used wild type, RAGE null, and lung‐specific RAGE overexpressing transgenic (TG) mice and evaluated the elaboration of inflammatory mediators in bronchoalveolar lavage fluid (BALF). Select mice were administered semi‐synthetic glycosaminoglycan ethers (SAGEs), a family of anionic, partially lipophilic sulfated polysaccharide derivatives known to inhibit RAGE signaling. Mice were exposed to room air (RA) or SHS from three Kentucky 3R4F research cigarettes via a nose‐only delivery system (Sireq Scientific, Montreal, Canada) five days a week and ip injections of PBS or SAGE (a 30mg/kg body weight) occurred three times per week from PN40 until sacrifice date on PN70. RAGE mRNA and protein expression was elevated in wild type and TG mice following SHS exposure and no expression was detected in RAGE nulls. BALF analyses revealed RAGE‐mediated control of leukocyte extravasation and a multiplex cytokine array confirmed a role for RAGE in the coordination of pro‐inflammatory chemokine/cytokine secretion. Among other mediators, TNF‐a, MIP‐2, and IL‐1b were each differentially secreted by lung tissue following SHS exposure and concentrations were significantly decreased in BALF from exposed RAGE null or wild type mice concomitantly administered SAGEs. In summary, inflammatory responses induced by SHS exposure were influenced by the availability of RAGE, as evidenced by RAGE nulls and SAGE treatment. These data reveal fascinating data suggesting the utility of RAGE abrogation in lessening smoke‐induced pulmonary exacerbations.

RAGE signaling during tobacco smoke-induced lung inflammation and potential therapeutic utility of SAGEs

BackgroundSmoke exposure culminates as a progressive lung complication involving airway inflammation and remodeling. While primary smoke poses the greatest risk, nearly half of the US population is also at risk due to exposure to secondhand smoke (SHS).MethodsWe used WT, RAGE−/− (KO), and Tet-inducible lung-specific RAGE overexpressing transgenic (TG) mice to study the role of RAGE during short-term responses to SHS. We evaluated SHS effects in mice with and without semi-synthetic glycosaminoglycan ethers (SAGEs), which are anionic, partially lipophilic sulfated polysaccharide derivatives known to inhibit RAGE signaling. TG Mice were weaned and fed doxycycline to induce RAGE at postnatal day (PN) 30. At PN40, mice from each line were exposed to room air (RA) or SHS from three Kentucky 3R4F research cigarettes via a nose-only delivery system (Scireq Scientific, Montreal, Canada) five days a week and i.p. injections of PBS or SAGE (30 mg/kg body weight) occurred three times per week from PN40-70 before mice were sacrificed on PN70.ResultsRAGE mRNA and protein expression was elevated following SHS exposure of control and TG mice and not detected in RAGE KO mice. Bronchoalveolar lavage fluid (BALF) analysis revealed RAGE-mediated influence on inflammatory cell diapedesis, total protein, and pro-inflammatory mediators following exposure. Lung histological assessment revealed indistinguishable morphology following exposure, yet parenchymal apoptosis was increased. Inflammatory signaling intermediates such as Ras and NF-κB, as well as downstream responses were influenced by the availability of RAGE, as evidenced by RAGE KO and SAGE treatment.ConclusionsThese data provide fascinating insight suggesting therapeutic potential for the use of RAGE inhibitors in lungs exposed to SHS smoke.

Comparative transcriptomics in human COPD reveals dysregulated genes uniquely expressed in ferrets

BackgroundChronic obstructive pulmonary disease (COPD) is a progressive lung disease with poor treatment options. However, most mouse models of COPD produce a primarily emphysematous disease not recapitulating clinically meaningful COPD features like chronic bronchitis.MethodsWild-type ferrets (Mustela putorius furo) were divided randomly into two groups: whole body cigarette smoke exposure and air controls. Ferrets were exposed to smoke from 1R6F research cigarettes, twice daily for six months. RNA-sequencing was performed on RNA isolated from lung tissue. Comparative transcriptomics analyses of COPD in ferrets, mice, and humans were done to find the uniquely expressed genes. Further, Real-time PCR was performed to confirmed RNA-Seq data on multiple selected genes.ResultsRNA-sequence analysis identified 420 differentially expressed genes (DEGs) that were associated with the development of COPD in ferrets. By comparative analysis, we identified 25 DEGs that are uniquely expressed in ferrets and humans, but not mice. Among DEGs, a number were related to mucociliary clearance (NEK-6, HAS1, and KL), while others have been correlated with abnormal lung function (IL-18), inflammation (TREM1, CTSB), or oxidative stress (SRX1, AHRR). Multiple cellular pathways were aberrantly altered in the COPD ferret model, including pathways associated with COPD pathogenesis in humans. Validation of these selected unique DEGs using real-time PCR demonstrated > absolute 2-fold changes in mRNA versus air controls, consistent with RNA-seq analysis.ConclusionCigarette smoke-induced COPD in ferrets modulates gene expression consistent with human COPD and suggests that the ferret model may be uniquely well suited for the study of aspects of the disease.