This is the first study to report changes in nicotine delivery and biomarkers of tobacco exposure following a short-term product switch from CCs to either an EC or NG in a controlled environment. The study shows that nicotine exposure decreased in both groups but was maintained closer to CC smoking with the EC groups. Biomarkers of tobacco combustion decreased to similar levels in both EC and gum groups.

a b s t r a c tTobacco toxicant-related exposure reduction is an important tool in harm reduction. Cigarette per day reduction (CPDR) occurs as smokers migrate from smoking cigarettes to using alternative tobacco/nicotine products, or quit smoking. Few reports characterize the dose-response relationships between CPDR and effects on exposure biomarkers, especially at the low end of CPD exposure (e.g., 5 CPD). We present data on CPDR by characterizing magnitudes of biomarker reductions. We present data from a wellcontrolled, one-week clinical confinement study in healthy smokers who were switched from smoking 19-25 CPD to smoking 20, 10, 5 or 0 CPD. Biomarkers were measured in blood, plasma, urine, and breath, and included smoke-related toxicants, urine mutagenicity, smoked cigarette filter analyses (mouth level exposure), and vital signs. Many of the biomarkers (e.g., plasma nicotine) showed strong CPDR doseresponse reductions, while others (e.g., plasma thiocyanate) showed weaker dose-response reductions. Factors that lead to lower biomarker reductions include non-CPD related contributors to the measured response (e.g., other exposure sources from environment, life style, occupation; inter-individual variability). This study confirms CPDR dose-responsive biomarkers and suggests that a one-week design is appropriate for characterizing exposure reductions when smokers switch from cigarettes to new tobacco products.

Existing US epidemiological data demonstrate that consumption of smokeless tobacco, particularly moist snuff, is less harmful than cigarette smoking. However, the molecular and biochemical changes due to moist snuff consumption relative to smoking remain incompletely understood. We previously reported that smokers (SMK) exhibit elevated oxidative stress and inflammation relative to moist snuff consumers (MSC) and non-tobacco consumers (NTC), based on metabolomic profiling data of saliva, plasma, and urine from MSC, SMK, and NTC. In this study, we investigated the effects of tobacco consumption on additional metabolic pathways using pathway-based analysis tools. To this end, metabolic pathway enrichment analysis and topology analysis were performed through pair-wise comparisons of global metabolomic profiles of SMK, MSC, and NTC. The analyses identified >8 significantly perturbed metabolic pathways in SMK compared with NTC and MSC in all 3 matrices. Among these differentially enriched pathways, perturbations of caffeine metabolism, energy metabolism, and arginine metabolism were mostly observed. In comparison, fewer enriched metabolic pathways were identified in MSC compared with NTC (5 in plasma, none in urine and saliva). This is consistent with our transcriptomics profiling results that show no significant differences in peripheral blood mononuclear cell gene expression between MSC and NTC. These findings, taken together with our previous biochemical, metabolomic, and transcriptomic analysis results, provide a better understanding of the relative changes in healthy tobacco consumers, and demonstrate that chronic cigarette smoking, relative to the use of smokeless tobacco, results in more pronounced biological changes, which could culminate in smoking-related diseases.

Background: The existing US epidemiological data show that long-term cigarette smokers are at higher risk of developing serious diseases relative to moist snuff consumers. To understand the effects of tobacco consumption, global metabolomic profiles were generated. Here, we describe metabolomic changes in oxidative stress and inflammation pathways. Methods: Matching plasma, urine, and saliva samples from chronic/long-term smokers (SMK), moist snuff consumers (MSC), and non-tobacco consumers (NTC), 40 subjects in each cohort, were collected in a crosssectional biomarker discovery study. Untargeted metabolomics and data analyses were performed using Metabolon's proprietary technology. Results: Several biochemicals that significantly differed between study cohorts were identified in all three matrices, with most metabolites found in urine. Random forest analyses of the metabolomes grouped study subjects with a high accuracy and indicated that nicotine and its metabolites primarily drive separation between the NTC and MSC; otherwise, metabolomic profiles of NTC and MSC are more similar to each other, and SMK appear to manifest a distinct metabolomic profile. SMK exhibit lower levels of antioxidants, changes in glutathione metabolism and purine degradation pathways, docosahexaenoate, arachidonate, and 12-hydroxyeicosatetraenoic acid, suggesting increased oxidative stress and inflammation relative to MSC and NTC. Conclusions: Metabolomic profiles show that while SMK and MSC cohorts exhibit higher levels of nicotine and its metabolites, SMK manifest evidence of increased oxidative stress and inflammation relative to MSC and NTC. These observed biochemical changes in the SMK could be likely due to the combustion-related toxicants present in cigarette smoke. Impact: Several differentiating metabolites identified herein could be utilized as potential biomarkers of effect. Further, the metabolomic profiles improve our understanding of biological changes in tobacco consumers.

Cigarette smoking is a major risk factor for several human diseases. Chronic inflammation, resulting from increased oxidative stress, has been suggested as a mechanism that contributes to the increased susceptibility of smokers to cancer and microbial infections. We have previously shown that whole-smoke conditioned medium (WS-CM) and total particulate matter (TPM) prepared from Kentucky 3R4F reference cigarettes [collectively called as combustible tobacco product preparations (TPPs)] potently suppressed agonist-stimulated cytokine secretion and target cell killing in peripheral blood mononuclear cells (PBMCs). Here we have investigated the role of oxidative stress from TPPs, which alters inflammatory responses in vitro. Particularly, we investigated the mechanisms of WS-CM-induced suppression of select cytokine secretions in Toll-like receptor (TLR) agonist-stimulated cells and target cell killing by effector cells in PBMCs. Pretreatment with N-acetyl cysteine (NAC), a precursor of reduced glutathione and an established anti-oxidant, protected against DNA damage and cytotoxicity caused by exposure to WS-CM. Similarly, secretion of tumor necrosis factor (TNF), interleukin (IL)-6, and IL-8 in response to TLR-4 stimulation was restored by pretreatment with NAC. Target cell killing, a functional measure of cytolytic cells in PBMCs, is suppressed by WS-CM. Pretreatment with NAC restored the target cell killing in WS-CM treated PBMCs. This was accompanied by higher perforin levels in the effector cell populations. Collectively, these data suggest that reducing oxidative stress caused by cigarette smoke components restores select immune responses in this ex vivo model.