G.L. Prasad

Evaluation of cytotoxicity of different tobacco product preparations.

Acute exposure to cigarette smoke or its components triggers diverse cellular effects, including cytotoxicity. However, available data regarding the potential cytotoxic effects of smokeless tobacco (ST) extracts lack consensus. Here, we investigated the relative biological effects of 2S3 reference ST, and whether ST elicits differential cellular/molecular responses compared to combustible tobacco product preparations (TPPs) prepared from 3R4F cigarettes. Total particulate matter (TPM) and whole smoke conditioned medium (WS-CM) were employed as combustible TPPs, while the ST extract was used as non-combustible TPP. HL60, THP1 cells and human PBMCs were used to examine the effects of TPPs in short-term cell culture. Corresponding EC(50) values, normalized for nicotine content of the TPPs, suggest that combustible TPPs induced higher cytotoxicity as follows: WS-CM TPM ≥ ≫ST extract>nicotine. While all three TPPs induced detectable levels of DNA damage and IL8 secretion, the combustible TPPs were significantly more potent than the ST preparation. The major PBMC subsets showed differential cytotoxicity to combustible TPPs as follows: CD4>CD8>monocytes>NK cells. These findings suggest that, relative cytotoxic and other cell biological effects of TPPs are dose-dependent, and that ST extract is the least cytotoxic TPP tested in this study.

Combustible and non-combustible tobacco product preparations differentially regulate human peripheral blood mononuclear cell functions

Natural killer (NK) cells and T cells play essential roles in innate and adaptive immune responses in protecting against microbial infections and in tumor surveillance. Although evidence suggests that smoking causes immunosuppression, there is limited information whether the use of smokeless tobacco (ST) products affects immune responses. In this study, we assessed the effects of two preparations of cigarette smoke, ST extract and nicotine on T cell and NK cell responses using Toll-like receptor-ligand stimulated human peripheral blood mononuclear cells (PBMCs). The tobacco product preparations (TPPs) tested included whole smoke conditioned media (WS-CM), total particulate matter (TPM) and a ST product preparation in complete artificial saliva (ST/CAS). The PBMCs were stimulated with polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS). A marked reduction of the expression of intracellular IFN-γ and TNF-α was evident in NK cells and T cells treated with WS-CM and TPM. Consistently, attenuation of ligand-induced secretion of cytokines (IL-1β, IL-10, IL-12 and TNF-α) from PBMCs treated with WS-CM and TPM were observed. While the treatment with TPPs did not alter the expression of the maturation marker CD69, WS-CM and TPM inhibited the cytolytic activity of human PBMCs. Suppression of perforin by WS-CM was also detected. Although interference from the vehicle confounded the interpretation of effects of ST/CAS, some effects were evident only at high concentrations. Nicotine treatment minimally impacted expression of cytokines and cytolytic activity. Data presented herein suggests that the function of NK cells and T cells is influenced by exposure to TPPs (based on equi-nicotine units) in the following order: WS-CM>TPM>ST/CAS. These findings are consistent with the hypothesis put forward by others that chronic smoking leads to immunosuppression, an effect that may contribute to increased microbial infections and cancer incidence among smokers.

A cross-sectional study of biomarkers of exposure and effect in smokers and moist snuff consumers.

Abstract Background: Cigarette smoking is a major risk factor for several chronic diseases. Epidemiological data indicate the use of smokeless tobacco (ST) is associated with significantly lower risk for smoking-related diseases compared to cigarettes. Several biomarkers of exposure (BioExp) and effect (BioEff) associated with smoking and use of moist snuff (ST) were evaluated. Methods: A single site, cross-sectional clinical study enrolled three groups of generally healthy male smokers (SMK), moist snuff consumers (MSC), and non-tobacco consumers (NTC), and several BioExp and BioEff were evaluated. Results: Blood and urinary BioExp, including total nicotine equivalents and tobacco-specific nitrosamines, were higher in MSC compared to SMK. Biomarkers of combustion-related toxicants and cadmium were elevated in SMK. Elevated levels of some BioEff associated with oxidative stress (urinary isoprostanes and leukotriene E4), inflammation (white blood cell count), platelet activation (thromboxane metabolites), and lipid metabolism (apolipoprotein B100 and oxidized low-density lipoprotein) were observed in SMK relative to NTC and MSC (all p<0.05). The non-smoking groups (MSC and NTC) showed similar levels of combustion-related BioExp and BioEff. Conclusions: Higher levels of exposure to nicotine and some N′-nitrosamines may be observed in MSC, and SMK are exposed to higher levels of combustion-related toxicants. Changes in BioEff consistent with some aspects of inflammation, oxidative stress, and altered lipid metabolism were detected in SMK compared to the non-smoking groups. The biomarker data further improve our understanding of pathophysiological changes and the risk continuum associated with various tobacco products, and could be useful components of future assessments of tobacco products.

Gene expression profiles associated with cigarette smoking and moist snuff consumption

BackgroundAmong the different tobacco products that are available on the US market, cigarette smoking is shown to be the most harmful and the effects of cigarette smoking have been well studied. US epidemiological studies indicate that non-combustible tobacco products are less harmful than smoking and yet very limited biological and mechanistic information is available on the effects of these alternative tobacco products. For the first time, we characterized gene expression profiling in PBMCs from moist snuff consumers (MSC), compared with that from consumers of cigarettes (SMK) and non-tobacco consumers (NTC).ResultsMicroarray analysis identified 100 differentially expressed genes (DEGs) between the SMK and NTC groups and 46 DEGs between SMK and MSC groups. However, we found no significant differences in gene expression between MSC and NTC. Both hierarchical clustering and principle component analysis revealed that MSC and NTC expression profiles were more similar than to SMK. Random forest classification identified a subset of DEGs which predicted SMK from either NTC or MSC with high accuracy (AUC 0.98).ConclusionsPMBC gene expression profiles of NTC and MSC are similar to each other, while SMK exhibit distinct profiles with alterations in immune related pathways. In addition to discovering several biomarkers, these studies support further understanding of the biological effects of different tobacco products.Trial registrationClinicalTrials.gov. Identifier: NCT01923402. Date of Registration: August 14, 2013. Study was retrospectively registered.

Role of Oxidative Stress in the Suppression of Immune Responses in Peripheral Blood Mononuclear Cells Exposed to Combustible Tobacco Product Preparation

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.

Inflammatory profile analysis reveals differences in cytokine expression between smokers, moist snuff users, and dual users compared to non-tobacco consumers

Objective and design The aim of this study is to investigate the inflammatory alterations due to the use of smokeless tobacco and dual use of smokeless tobacco and cigarettes, relative to smoking. Subjects Plasma and saliva samples were collected from healthy smokers (SMK‐100 subjects), moist snuff users (MSC‐89 subjects), the dual users (DUSMK‐49 subjects), and non‐tobacco consumers (NTC‐99 subjects) from two cross‐sectional studies. Methods Luminex Human InflammationMAP® 1.0 panel, a multiplex immunoassay. Results SMK and DUSMK exhibited larger number of alterations in the expression of inflammatory analytes compared to NTC. Eight analytes were significantly elevated (p ≤ .05) within plasma samples of SMK compared to NTC, while one 1 analyte was elevated between the MSC and NTC groups. DUSMK exhibited different levels of 11 analytes, relative to NTC. MSC displayed fewer alterations in inflammatory protein expression compared to smoker groups, and the inflammatory profile of MSC resembles NTC. Five analytes (ICAM‐1, VEGF, MMP‐9, ferritin and fibrinogen) emerged as potential biomarkers distinguishing tobacco consumers (p < .02). Conclusions We identified a set of five proteins as potential biomarkers that can inform of inflammation status due to tobacco usage. Our findings contribute a better understanding of how the use of different tobacco products contributes to inflammation.

Methods to evaluate cytotoxicity and immunosuppression of combustible tobacco product preparations.

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-γ, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-γ, TNF-α, and MIP-1α were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts.

Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1h or 5h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5h), which was increased by nicotine but suppressed by other components of STE. Within 2h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research.