Cigarette smoking is associated with impairment of repair mechanisms essential for vascular endothelium homeostasis. Reducing the exposure to smoke toxicants may result in the mitigation of the harmful effect on the endothelium and cardiovascular disease development. Previous investigations performed by the tobacco industries evaluated in vitro the effect of electronic cigarette (e-cig) compared to cigarette smoke demonstrating a significant reduction in endothelial cell migration inhibition following e-cig aerosol exposure. In the present study, we replicated one of these studies, evaluating the effects of cigarette smoke on endothelial cell migration compared to an e-cig, adding to the comparison two heated tobacco products (THPs). We performed an in vitro scratch wound assay on endothelial cells with a multi-center approach (ring-study) to verify the robustness and reliability of the results obtained in the replicated study. Consistently with the original study, we observed a substantial reduction of the effects of e-cig on endothelial cell migration compared to cigarette smoke. This reduction was observed with the exposure of endothelial cells to the extracts from THPs. Cigarette smoking reduced endothelial wound healing capability already at low concentrations (12.5%) and in a concentration-dependent manner, e-cig and HTPs aerosol showed no effect on endothelial cells until 80%-100% concentration. In conclusion, our study further confirms the importance of e-cig and THPs as a possible harm reduction strategy for cardiovascular diseases development in smokers.

AUTHORS: Massimo Caruso^1,2, Rosalia Emma¹, Alfio Distefano¹, Sonja Rust³, Konstantinos Poulas^4,5, Antonio Giordano⁶, Vladislav Volarevic⁷, Konstantinos Mesiakaris^4,5, Silvia Boffo⁶, Aleksandar Arsenijevic⁷, Georgios Karanasios^4,5, Roberta Pulvirenti¹, Aleksandar Ilic⁷, Angelo Canciello⁶, Pietro Zuccarello⁸, Margherita Ferrante⁸, Riccardo Polosa^2,3,9, Giovanni Li Volti^1,2 (Replica Project Group)

AFFILIATIONS: ¹Department of Biomedical and Biotechnological Sciences, University of Catania, Italy || ²Center of Excellence for the Acceleration of Harm Reduction (CoEHAR), University of Catania, Italy || ³ECLAT Srl, spin off of the University of Catania, Italy || ⁴Institute for Research and Innovation, IRIS, Patras Science Park, Patras, Greece || ⁵Laboratory of Molecular Biology and Immunology, Department of Pharmacy, University of Patras, Greece || ⁶Sbarro Institute for Cancer Research and Molecular Medicine, Department of Biology, College of Science and Technology, Temple University, Philadelphia, USA || ⁷Center for Molecular Medicine and Stem Cell Research, Department of Microbiology and Immunology, Faculty of Medical Sciences, University of Kragujevac, Serbia || ⁸Department of Medical, Surgical Sciences and Advanced Technologies “G.F. Ingrassia”, University of Catania, Italy || ⁹Department of Clinical and Experimental Medicine, University of Catania, Italy

Tobacco heating system (THS) products are intended to be a substitution product for conventional cigarette for smokers attempting to quit smoking. Tobacco products health risks are strongly correlated with the presence of harmful and potentially harmful constituents (HPHCs) within the product. The work was conducted following common steps in the risk assessment of chemicals, which includes hazard identification, hazard characterization, exposure assessment, and risk characterization. However, all hazard identification and characterization data, as well as quantification data of HPHCs in THS aerosol, were based on the literature study, whereas quantification data of HPHCs in kretek cigarette were based on our results. By comparing the HPHCs concentration and their hazard characterization in both the generated aerosol of THS product and smoke of kretek cigarette, as well as their exposure to consumer, health risk profile comparison of the two products is possible. Our results revealed that THS product showed lower health risk profile compared to kretek cigarette.

AUTHORS: Rahmana-Emran Kartasasmita, Daryono-Hadi Tjahjono, Fransiska Kurniawan, Tasia Amelia, Aderian-Novito Setiawan

AFFILIATION: School of Pharmacy, Bandung Institute of Technology, Indonesia

The harm caused by cigarette smoking is overwhelmingly due to inhalation of products of combustion. For that reason, Electronic Nicotine Delivery Systems (ENDS), which deliver nicotine, but without combustion, are considered an important tool for tobacco harm reduction. Exposure to smoking-related toxicants can be measured using biomarkers of exposure (BOEs). This analysis uses biomarker data from wave 5 of the PATH Study of US adults (2018-19), analyzing creatinine-adjusted urinary BOEs for nicotine, 3 heavy metals, and 14 smoking-related volatile organic compounds (VOCs) (e.g., mandelic acid, a marker of styrene exposure, related to cancer; HPMA, a marker of exposure to acrolein, a respiratory and cardiovascular toxicant). Analyses compared BOE levels in 151 exclusive ENDS users to those in 1,341 exclusive cigarette smokers and in 1,846 past 30-day non-users of tobacco; BOEs in 115 dual users (cigarettes and ENDS) were also compared to smokers. As expected, markers of nicotine exposure in ENDS users and dual users did not differ from those in smokers. Except for one (PMA, a marker for benzene exposure) all other BOEs were significantly higher in smokers vs non-users, making them potentially informative. All of these BOEs, representing 13 VOCs and 3 heavy metals, were significantly lower in ENDS users than in smokers. On 11 of these BOEs, levels in ENDS users were also not significantly different than those in non-tobacco users. Dual users, who reported smoking an average of 10.3 [95% CI: 8.5,12.2] cigarettes per day, showed BOE levels intermediate between smokers’ and ENDS users’, consistently lower than smokers’, significantly so for 6 of the BOEs. These findings in a real-world representative US population confirm and extend findings from other studies showing that use of ENDS is associated with exposure to much lower levels of many harmful and potentially harmful chemicals associated with smoking-related disease. This further reinforces the potential of ENDS for tobacco harm reduction among smokers.

AUTHORS: Nathan M. Holt¹, Saul Shiffman², Ryan A. Black¹, Nicholas I. Goldenson¹, Mark A. Sembower², Michael J. Oldham¹

AFFILIATIONS: ¹Juul Labs, Inc. || ²Pinney Associates, Inc.

Disclosures: The analysis was sponsored by Juul Labs, Inc. Authors Holt, Black, Goldenson, and Oldham are employed by Juul Labs, Inc. Through Pinney Associates, authors Shiffman and Sembower provide consulting on tobacco harm reduction to Juul Labs, Inc. on an exclusive basis.

Nicotine has been described as the main component of cigarette which is responsible for a wide variety of neurochemical and behavioral effects. The aims of the present study were to investigate the effects of nicotine intake on behavior parameters (anxiety-like behavior, mobility) acetylcholinesterase’s (AChE) isoforms (G1, G4) activity on specific brain regions (cerebral hemispheres, cerebellum) of adult male mice.

Mice were divided into 4 groups: control, 1 cigarette’, 2 cigarettes’ and Tobacco Heating System 2.2 (THS2.2’) smoke/aerosol exposed groups. The exposure was carried out in a unique smoking device with whole body exposure in smoke. The behavioral analysis was assessed by using the open field test (measurements at 2 time point: 5min and 10min). The activity of G1 and G4 AChE’s isoforms was determined by using Ellman’s colorimetric method.

The results show that no change has been found in both anxiety and mobility after smoke exposure of 1 cigarette.Mice exposed to smoke of 2 cigarettes exhibited increased anxiety levels and decreased mobility at the 10min time point. Moreover, mice exposed to THS2.2 aerosol exhibited decreased anxiety levels only at 5min time point and increased mobility in both time points. Furthermore, all the exposures reduce AChE’s activity (both isoforms) in cerebral hemispheres. The exposure of 2 cigarette and THS2.2’ aerosol caused reduction of G1 isoform’s activity and the exposure of 1 and 2 cigarette smoke caused reduction of activity of G4 isoform in cerebellum.

AUTHORS: Korina Atsopardi^1,2, Konstantinos Mesiakaris¹, Ioannis Plastourgos², Marigoula Margarity², Konstantinos Poulas¹

AFFILIATIONS: ¹Laboratory of Molecular Biology and Immunology, Department of Pharmacy, University of Patras, Rio, Greece || ²Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Rio, Greece

Global scientific communities and government agencies recognized the Ames assay as an initial screen for the assessment of mutagenicity, therefore it is used to determine the mutagenic potential of chemical products, including cigarette smoke and modified risk products (MRPs) vapor. MRPs indicate the alternative products to cigarette smoke, able to deliver nicotine without smoke, such as electronic cigarette (e-cig) and tobacco heating products (THPs). Unlike conventional cigarettes, MRPs seem to exhibit reduced health risks and can help smokers to quit smoking. Previous studies on mutagenic effect of MRPs by the tobacco industries showed reduced mutagenicity compared to cigarette smoke. In the present study, we replicated one of these studies, assessing the mutagenicity effect of Myblu e-cig vapor on TA98 and TA100 Salmonella typhimurium strains with and without S9 enzymatic activation. 1R6F cigarette whole smoke (from 1 to 5 cigarettes, 9 puffs each, following HCI regime) and Myblu whole aerosol (from 60 to 300 puffs following CRM81 regime) were bubbled through 10 ml of PBS bacterial suspensions. Bubbling with filtered room air was used as negative control. Dose response curve of 1R6F smoke and Myblu aerosol mutagenic activities were calculated with fold changes in the number of revertants on the treated plates and the untreated controls, and tested for significance. Consistently with the original study, we observed significant dose-depended increase of revertant number for both TA98 and TA100 after exposure to 1R6F whole smoke, either with or without S9 enzymatic activation. Instead, the Myblu e-cig aerosol (up to 300 puffs) did not increase the number of revertants compared with the Air negative control in either strains with and without S9 enzymatic activation. In conclusion, we confirmed the reduced mutagenicity of e-cig aerosol compared to cigarette smoke, confirming that e-cigs can be a valuable tool for the harm reduction strategies in smokers.

AUTHORS: Massimo Caruso^1,2, Virginia Fuochi¹, Rosalia Emma¹, Sonja Rust³, Alfio Distefano¹, Riccardo Polosa^2,3,4, Giovanni Li Volti^1,2

AFFILIATIONS: ¹Department of Biomedical and Biotechnological Sciences, University of Catania, Italy || ²Center of Excellence for the Acceleration of Harm Reduction (CoEHAR), University of Catania, Italy || ³ECLAT Srl, spin off of the University of Catania, Italy || ⁴Department of Clinical and Experimental Medicine, University of Catania, Italy

Background: Fagerstrom score is a validated marker of nicotine addiction in smokers.

Material and Methods: In a prospective study, we investigated a) the predictive value of Fagerstrom score for the smoking status in patients early after acute myocardial infarction (AMI) and b) the effectiveness of medically assisted smoking cessation programs in the prevention of relapsing to smoking post discharge. In 103 smokers (58±12 years, 79.6% males), we assessed Fagerstrom score during hospitalization for AMI. Patients filled a dedicated questionnaire including data on family, marital and educational status, habits related to smoking and were followed-up at 3 and 6 months after discharge.

Results: Twenty-eight patients (27.2%) did not quit smoking throughout the 6-months follow-up period (Fagerstrom score: 8.1±1.6), 39 patients (37.8%) ceased smoking at 3 months but relapsed to smoking at 6 months (score: 6.8±2.1), and only 34 patients (33%) had ceased smoking for 6 consecutive months (score: 5.2±2, p<0.05 for all comparisons between subgroups). By multivariate analysis, Fagerstrom score remained a significant predictor of smoking cessation at 6 months (OR: 0.72, 95% CI: 0.60-0.86, p<0.001). Out of 73 patients who abstained from smoking for the first 3 months post-AMI, those who participated in a smoking cessation program displayed lower rate of relapsing to smoking compared with those who opted to cease smoking without any support (33.3% vs 61.8%, p=0.012). Conclusions: Fagerstrom score is a useful predictor of smoking cessation 6 months post-AMI. Patients participating in a smoking cessation program display lower relapse rates post-discharge suggesting the need of well-organized smoking cessation clinics for secondary prevention of heart disease.

AUTHORS: Ignatios Ikonomidis¹, John Thymis¹, Kallirhoe Kourea¹, Gavriella Kostelli¹, Antria Neocleous¹, Konstantinos Katogiannis¹, George Makavos¹, Eftihia Polyzogopoulou², Panagiotis Plotas³, Vaia Lambadiari³, John Parissis²

AFFILIATIONS: ¹2^nd Cardiology Department, Attikon University Hospital, Medical School, National and Kapodistrian University of Athens, Greece || ²Emergency Medicine Department, Attikon University Hospital, National and Kapodistrian University of Athens, Greece || ³2^nd Department of Internal Medicine, Research Unit and Diabetes Centre, Attikon University Hospital, Medical School, National and Kapodistrian University of Athens, Greece