Pilot study to determine differences in breath odour between cigarette and e-cigarette consumers.

Cigarette smoke is known to influence breath odour, but the effect of e-cigarettes is unknown. In this pilot study, we aimed to determine differences in breath odour between cigarette smokers (CSs) and e-cigarette consumers (ECs) in 33 healthy subjects: 11 CSs, 11 ECs and 11 non-smokers (NSs). Breath was sampled at baseline and following product use (CSs and ECs) or a waiting period (NSs) by eight trained odour judges using a six-point smoke intensity scale and a nine-point hedonic scale. We observed a statistically significant difference between CSs and ECs. Smoke intensity values were significantly higher in CSs than ECs and NSs, which were comparable both at baseline and after product use. In addition, hedonic values for CSs were significantly lower than both NSs and ECs after product use. These acute results indicate that cigarette and e-cigarette use results in significantly different breath. ECs breath has a reduced smoke odour and more pleasant aroma than CSs, and is comparable to NSs. This suggests there may be cosmetic benefits for CSs who quit smoking or switch to exclusive use of ECs. Further studies are required to understand the long-term effects of e-cigarette use on breath odour.

A novel clinical method to measure skin staining reveals activation of skin damage pathways by cigarette smoke.

BACKGROUND: Long-term use of cigarettes can result in localised staining and aging of smokers' skin. The use of tobacco heating products (THPs) and electronic cigarettes (ECs) has grown on a global scale; however, the long-term effect of these products' aerosols on consumers' skin is unknown. This pilot clinical study aimed to determine whether THP or EC aerosol exposure results in skin staining or activation of biomarkers associated with oxidative stress. MATERIALS AND METHODS: Eight areas were identified on the backs of 10 subjects. Two areas were used for air control, and two areas exposed to 32-puffs of cigarette smoke (CS), THP or EC aerosols, which were delivered to the skin using a 3-cm diameter exposure chamber and smoke engine. Skin colour was measured using a Chromameter. Squalene (SQ), SQ monohydroperoxide (SQOOH) and malondialdehyde (MDA) levels were measured in sebum samples by mass spectrometry and catalase colorimetry. RESULTS: CS exposure significantly increased skin staining, SQOOH and MDA levels and SQOOH/SQ ratio. THP and EC values were significantly lower than CS; EC values being comparable to air control. THP values were comparable to EC and air control at all endpoints, apart from skin staining. SQ and catalase levels did not change with exposure. CONCLUSIONS: CS stained skin and activated pathways known to be associated with skin damage. THPs and ECs produced significantly lower values, suggesting they could offer hygiene and cosmetic benefits for consumers who switch exclusively from smoking cigarettes. Further studies are required to assess longer-term effects of ECs and THPs on skin function.

Enamel staining with e-cigarettes, tobacco heating products and modern oral nicotine products compared with cigarettes and snus: An in vitro study.

PURPOSE: To evaluate the effect of cigarette smoke, smokeless tobacco (e.g. snus), tobacco heating products (THP), electronic cigarettes (EC), and modern oral nicotine products on tooth staining. METHODS: In this in vitro study, staining was assessed for 86 days following exposure of bovine enamel samples to a scientific reference cigarette (1R6F), a THP (glo), an EC (ePen 3), a reference snus product (CRP1.1), and a modern oral product (LYFT). Red wine and coffee were used as positive controls and DMSO and complete artificial saliva as negative controls. Whether brushing could reduce staining levels was also assessed. Changes in staining levels were assessed using the Commission Internationale de L'éclairage L*a*b* method. RESULTS: Enamel staining increased with incubation time, and cigarette smoke, snus, coffee and wine induced statistically higher staining levels. THP, EC and modern oral exposure induced minimal staining levels that were also comparable to negative control samples. At day 86, ΔE mean and SD values were 28.50 ± 3.14, 19.76 ± 1.26, 17.35 ± 3.44, 16.22 ± 2.07, 18.30 ± 3.82, 4.10 ± 1.99, 11.30 ± 2.60, 49.56 ± 2.44 for cigarette, glo, EC with blended tobacco, EC with rich tobacco, reference snus product, modern oral product, coffee or wine. The control ΔE mean and SD values at day 86 were 18.68 ± 3.89 for DMSO and 2.17± 0.78 for complete artificial saliva. The ΔE values for all DMSO extracted samples and control increased from day 1 to 86, which suggests that the DMSO used to extract the samples contributes to the enamel sample staining levels. Staining levels were reduced by brushing. CLINICAL SIGNIFICANCE: Cigarette smoke, red wine, snus and coffee stained enamel. Exposure to THP, EC or modern oral product extracts for 86 days resulted in minimal enamel staining. Further studies are required to assess the long-term impact on staining and the oral cavity following consumer exclusive use of EC, THP or modern oral products.

Development of a novel method to measure material surface staining by cigarette, e-cigarette or tobacco heating product aerosols.

Tobacco smoke (CS) may visually stain indoor surfaces including ceilings, walls and soft furnishings over time. Potentially reduced risk products (PRRPs) such as e-cigarettes (EC) and tobacco heating products (THP) produce chemically less complex aerosols with significantly reduced levels of toxicants, particles and odour. However, the potential effects of EC and THP aerosols on the staining of indoor surfaces are currently unknown. In this study, an exposure chamber was developed as a model system to enable the accelerated staining of wallpaper and cotton samples by a scientific reference cigarette (3R4F), three THP (glo™, glo™ pro, glo™ sens) and an e-cigarette (iSwitch Maxx). Exposure to 3R4F reference cigarettes caused the greatest level of staining, which was significantly higher than glo™, glo™ pro, glo™ sens or iSwitch Maxx aerosols, all of which showed relatively little colour change. Exposure to 200-1000 puffs of 3R4F cigarette smoke resulted in a visible dose response effect to wallpaper and cotton samples which was not observed following exposure to glo™, glo™ pro, glo™ sens or iSwitch Maxx aerosols. Aging of the samples for 4 weeks post-exposure resulted in changes to the staining levels, however PRRP staining levels were minimal and significantly lower than 3R4F exposed samples. For the first time, diverse PRRPs across the tobacco and nicotine products risk continuum have been assessed in vitro for their impact on surface staining. CS exposure significantly increased the level of wallpaper and cotton staining, whereas exposure to glo™, glo™ pro, glo™ sens or iSwitch Maxx aerosols resulted in significantly reduced levels of staining, staining levels were also comparable to untreated control samples.

Assessment of enamel discoloration in vitro following exposure to cigarette smoke and emissions from novel vapor and tobacco heating products.

PURPOSE: To evaluate in vitro enamel sample discoloration following exposure to a scientific reference cigarette (3R4F) or emissions from next generation tobacco and nicotine products (NGPs) such as electronic cigarettes (EC) and tobacco heating products (THP). METHODS: Bovine enamel blocks (6.5 × 6.5 mm) were prepared and pre-incubated with human or artificial saliva, to form a pellicle layer before exposure to either particulate matter (PM) or whole aerosols. PM was prepared by capturing 3R4F cigarette smoke (CS), a commercial THP (THP1.0) or a novel vapor product (NVP)/next generation e-cigarette aerosols on Cambridge filter pads followed by elution with dimethyl sulfoxide (DMSO). Ten enamel samples were exposed to each PM for 14 days. For aerosol exposure, 12 enamel samples were exposed (200 puffs per day, for 5 consecutive days) to 3R4F CS or THP1.0 and NVP aerosols. Control samples were incubated with DMSO (PM study) or phosphate buffered saline (PBS, aerosol study). Individual enamel sample color readings (L*, a*, b*) were measured at baseline and on each exposure day. Mean ΔL*, Δa*, Δb* and ΔE values were calculated for each product or control. A one-way ANOVA was used to assess the differences between the products and controls. The Tukey procedure for pairwise comparisons was also used. RESULTS: At all timepoints, 3R4F PM and CS induced enamel discoloration that was statistically significant (< 0.0001) when compared to THP1.0 or NVP. After 14-day PM exposure, mean ΔE values were 29.4± 3.6, 10.5 ± 2.3, 10.7 ± 2.6 and 12.6 ± 2.0 for 3R4F, THP1.0, NVP and DMSO control respectively. After 5-day CS or aerosol exposure, mean ΔE values were 26.2 ± 3.2, 3.6 ± 1.9, 3.4 ± 1.3, 5.3 ± 0.8 for 3R4F CS, THP1.0, NVP or PBS control, respectively. Both exposure methods demonstrated that THP1.0 and NVP induced minimal staining, mean ΔL* , Δa* , Δb* and ΔE values were comparable to DMSO or PBS controls. CLINICAL SIGNIFICANCE: For the first time, diverse NGPs across the risk continuum were assessed in vitro for their impact on enamel staining. CS exposure significantly increased the level of bovine enamel sample discoloration, whereas THP1.0 or NVP exposure resulted in values comparable to the controls.

Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing.

Many laboratories are working to develop in vitro models that will replace in vivo tests, but occasionally there remains a regulatory expectation of some in vivo testing. Historically, cigarettes have been tested in vivo for 90 days. Recently, methods to reduce and refine animal use have been explored. This study investigated the potential of reducing animal cigarette smoke (CS) exposure to 3 or 6 weeks, and the feasibility of separate lung lobes for histopathology or the Comet assay. Rats were exposed to sham air or CS (1 or 2 h) for 3 or 6 weeks. Respiratory tissues were processed for histopathological evaluation, and Alveolar type II cells (AEC II) isolated for the Comet assay. Blood was collected for Pig-a and micronucleus quantification. Histopathological analyses demonstrated exposure effects, which were generally dependent on CS dose (1 or 2 h, 5 days/week). Comet analysis identified that DNA damage increased in AEC II following 3 or 6 weeks CS exposure, and the level at 6 weeks was higher than 3 weeks. Pig-a mutation or micronucleus levels were not increased. In conclusion, this study showed that 3 weeks of CS exposure was sufficient to observe respiratory tract pathology and DNA damage in isolated AEC II. Differences between the 3 and 6 week data imply that DNA damage in the lung is cumulative. Reducing exposure time, plus analyzing separate lung lobes for DNA damage or histopathology, supports a strategy to reduce and refine animal use in tobacco product testing and is aligned to the 3Rs (replacement, reduction and refinement).

A comparative assessment of cigarette smoke aerosols using an in vitro air-liquid interface cytotoxicity test.

This study describes the evaluation of a modified air-liquid interface BALB/c 3T3 cytotoxicity method for the assessment of smoke aerosols in vitro. The functionality and applicability of this modified protocol was assessed by comparing the cytotoxicity profiles from eight different cigarettes. Three reference cigarettes, 1R5F, 3R4F and CORESTA Monitor 7 were used to put the data into perspective and five bespoke experimental products were manufactured, ensuring a balanced and controlled study. Manufactured cigarettes were matched for key variables such as nicotine delivery, puff number, pressure drop, ventilation, carbon monoxide, nicotine free dry particulate matter and blend, but significantly modified for vapor phase delivery, via the addition of two different types and quantities of adsorptive carbon. Specifically manufacturing products ensures comparisons can be made in a consistent manner and allows the research to ask targeted questions, without confounding product variables. The results demonstrate vapor-phase associated cytotoxic effects and clear differences between the products tested and their cytotoxic profiles. This study has further characterized the in vitro vapor phase biological response relationship and confirmed that the biological response is directly proportional to the amount of available vapor phase toxicants in cigarette smoke, when using a Vitrocell® VC 10 exposure system. This study further supports and strengthens the use of aerosol based exposure options for the appropriate analysis of cigarette smoke induced responses in vitro and may be especially beneficial when comparing aerosols generated from alternative tobacco aerosol products.

An improved method for the isolation of rat alveolar type II lung cells: Use in the Comet assay to determine DNA damage induced by cigarette smoke.

Smoking is a cause of serious diseases, including lung cancer, emphysema, chronic bronchitis and heart disease. DNA damage is thought to be one of the mechanisms by which cigarette smoke (CS) initiates disease in the lung. Indeed, CS induced DNA damage can be measured in vitro and in vivo. The potential of the Comet assay to measure DNA damage in isolated rat lung alveolar type II epithelial cells (AEC II) was explored as a means to include a genotoxicity end-point in rodent sub-chronic inhalation studies. In this study, published AEC II isolation methods were improved to yield viable cells suitable for use in the Comet assay. The improved method reduced the level of basal DNA damage and DNA repair in isolated AEC II. CS induced DNA damage could also be quantified in isolated cells following a single or 5 days CS exposure. In conclusion, the Comet assay has the potential to determine CS or other aerosol induced DNA damage in AEC II isolated from rodents used in sub-chronic inhalation studies.

The mutagenic assessment of mainstream cigarette smoke using the Ames assay: a multi-strain approach.

Salmonella typhimurium strains TA1535, TA1537, TA97, TA102 and TA104 were assessed for their suitability and use in conjunction with a Vitrocell(®) VC 10 Smoking Robot and 3R4F reference mainstream cigarette smoke. Little information exists on TA97, TA104, TA1535, TA1537 and TA102 using an aerosol 35mm spread-plate format. In this study, TA1535 and TA1537 were considered sub-optimal for use with a scaled-down format, due to low spontaneous revertant numbers (0-5 revertants/plate). In the context of a regulatory environment, TA97 is deemed an acceptable alternative for TA1537 and was therefore selected for whole smoke exposure in this study. However, there is no acceptable alternative for TA1535, therefore this strain was included for whole smoke exposure. TA1535, TA97, TA102 and TA104 were assessed for mutagenic responses following exposure to cigarette smoke at varying concentrations (using diluting airflow rates of 1.0, 4.0, 8.0 and 12.0L/min), and exposure times of 24 and 64min. A positive mutagenic response to cigarette smoke was observed in strain TA104 at both the 24 and 64min time points, in the presence of S-9, at the highest smoke concentration tested (1.0L/min diluting airflow). The three remaining strains were found to be unresponsive to cigarette smoke at all concentrations tested, in the presence and absence of metabolic activation. Cigarette smoke particulate deposition was quantified in situ of exposure using quartz crystal microbalance technology, enabling data to be presented against an associated gravimetric mass (µg/cm(2)). Finally, data obtained in this study were combined with previously published Ames data for TA98, TA100, YG1024, YG1042 and Escherichia coli (WP2 uvrA pKM101), generated using the same 35mm methodology. The combined data-set was used to propose an aerosol testing strategy, based on strain compatibility with the whole smoke aerosol, whilst maintaining the essence of the regulatory guidelines for the standard Ames assay.

The combination of two novel tobacco blends and filter technologies to reduce the in vitro genotoxicity and cytotoxicity of prototype cigarettes.

Tobacco smoke from a combustible cigarette contains more than 6000 constituents; approximately 150 of these are identified as toxicants. Technologies that modify the tobacco blend to reduce toxicant emissions have been developed. These include tobacco sheet substitute to dilute toxicants in smoke and blend treated tobacco to reduce the levels of nitrogenous precursors and some polyphenols. Filter additives to reduce gas (vapour) phase constituents have also been developed. In this study, both tobacco blend and filter technologies were combined into an experimental cigarette and smoked to International Organisation on Standardisation and Health Canada puffing parameters. The resulting particulate matter was subjected to a battery of in vitro genotoxicity and cytotoxicity assays - the Ames test, mouse lymphoma assay, the in vitro micronucleus test and the Neutral Red Uptake assay. The results indicate that cigarettes containing toxicant reducing technologies may be developed without observing new additional genotoxic hazards as assessed by the assays specified. In addition, reductions in bacterial mutagenicity and mammalian genotoxicity of the experimental cigarette were observed relative to the control cigarettes. There were no significant differences in cytotoxicity relative to the control cigarettes.

A method for assessment of the genotoxicity of mainstream cigarette-smoke by use of the bacterial reverse-mutation assay and an aerosol-based exposure system.

To date there are no widely accepted methods for the toxicological testing of complex gaseous mixtures and aerosols, such as cigarette smoke, although some modifications to the standard regulatory methods have been developed and used. Historically, routine testing of cigarettes has primarily focused on the particulate fraction of cigarette smoke. However, this fraction may not accurately reflect the full toxicity and mutagenicity of the smoke aerosol as a whole, which contains semi-volatiles and short-lived products of combustion. In this study we have used a modified version of the bacterial reverse-mutation (Ames) assay for the testing of mainstream smoke generated from 3R4F reference cigarettes with a Vitrocell(®) VC 10 exposure system. This method has been evaluated in four strains of Salmonella typhimurium (TA98, TA100, YG1024 and YG1042) and one strain of Escherichia coli (WP2 uvrA pKM101) in the absence and presence of a metabolic activation system. Following exposure at four concentrations of diluted mainstream cigarette-smoke, concentration-related and reproducible increases in the number of revertants were observed in all four Salmonella strains. E. coli strain WP2 uvrA pKM101 was unresponsive at the four concentrations tested. To quantify the exposure dose and to enable biological response to be plotted as a function of deposited mass, quartz-crystal microbalances were included in situ in the smoke-exposure set-up. This methodology was further assessed by comparing the responses of strain YG1042 to mainstream cigarette-smoke on a second VC 10 Smoking Robot. In summary, the Ames assay can be successfully modified to assess the toxicological impact of mainstream cigarette-smoke.

Development of a BALB/c 3T3 neutral red uptake cytotoxicity test using a mainstream cigarette smoke exposure system.

BACKGROUND: Tobacco smoke toxicity has traditionally been assessed using the particulate fraction under submerged culture conditions which omits the vapour phase elements from any subsequent analysis. Therefore, methodologies that assess the full interactions and complexities of tobacco smoke are required. Here we describe the adaption of a modified BALB/c 3T3 neutral red uptake (NRU) cytotoxicity test methodology, which is based on the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) protocol for in vitro acute toxicity testing. The methodology described takes into account the synergies of both the particulate and vapour phase of tobacco smoke. This is of particular importance as both phases have been independently shown to induce in vitro cellular cytotoxicity. FINDINGS: The findings from this study indicate that mainstream tobacco smoke and the gas vapour phase (GVP), generated using the Vitrocell® VC 10 smoke exposure system, have distinct and significantly different toxicity profiles. Within the system tested, mainstream tobacco smoke produced a dilution IC50 (dilution (L/min) at which 50% cytotoxicity is observed) of 6.02 L/min, whereas the GVP produced a dilution IC50 of 3.20 L/min. In addition, we also demonstrated significant dose-for-dose differences between mainstream cigarette smoke and the GVP fraction (P < 0.05). This demonstrates the importance of testing the entire tobacco smoke aerosol and not just the particulate fraction, as has been the historical preference. CONCLUSIONS: We have adapted the NRU methodology based on the ICCVAM protocol to capture the full interactions and complexities of tobacco smoke. This methodology could also be used to assess the performance of traditional cigarettes, blend and filter technologies, tobacco smoke fractions and individual test aerosols.

Characterisation of a Vitrocell® VC 10 in vitro smoke exposure system using dose tools and biological analysis.

BACKGROUND: The development of whole smoke exposure systems have been driven by the fact that traditional smoke exposure techniques are based on the particulate phase of tobacco smoke and not the complete smoke aerosol. To overcome these challenges in this study, we used a Vitrocell® VC 10 whole smoke exposure system. For characterisation purposes, we determined smoke deposition in relationship to airflow (L/min), regional smoke deposition within the linear exposure module, vapour phase dilution using a known smoke marker (carbon monoxide) and finally assessed biological responses using two independent biological systems, the Ames and Neutral Red uptake (NRU) assay. RESULTS: Smoke dilution correlates with particulate deposition (R2 = 0.97) and CO concentration (R2 = 0.98). Regional deposition analysis within the linear exposure chamber showed no statistical difference in deposited mass across the chamber at any airflows tested. Biological analysis showed consistent responses and positive correlations with deposited mass for both the Ames (R2 = 0.76) and NRU (R2 = 0.84) assays. CONCLUSIONS: We conclude that in our study, under the experimental conditions tested, the VC 10 can produce stable tobacco smoke dilutions, as demonstrated by particulate deposition, measured vapour phase smoke marker delivery and biological responses from two independent in vitro test systems.

Assessment of cigarette smoke particle deposition within the Vitrocell® exposure module using quartz crystal microbalances.

BACKGROUND: Cigarette smoking is a cause of a variety of serious diseases, and to understand the toxicological impact of tobacco smoke in vitro, whole smoke exposure systems can be used. One of the main challenges of the different whole smoke exposure systems that are commercially available is that they dilute and deliver smoke in different ways, limiting/restricting the cross-comparison of biological responses. This is where dosimetry - dose quantification - can play a key role in data comparison. Quartz crystal microbalance (QCM) technology has been put forward as one such tool to quantify smoke particle deposition in vitro, in real-time. RESULTS: Using four identical QCMs, installed into the Vitrocell® mammalian 6/4 CF Stainless exposure module, we were able to quantify deposited smoke particle deposition, generated and diluted by a Vitrocell® VC 10 Smoking Robot. At diluting airflows 0.5-4.0 L/min and vacuum flow rate 5 ml/min/well through the exposure module, mean particle deposition was in the range 8.65 ± 1.51 µg/cm(2)-0.72 ± 0.13 µg/cm(2). Additionally, the effect of varying vacuum flow rate on particle deposition was assessed from 5 ml/min/well - 100 ml/min/well. Mean deposited mass for all four airflows tested per vacuum decreased as vacuum rate was increased: mean deposition was 3.79, 2.75, 1.56 and 1.09 µg/cm(2) at vacuum rates of 5, 10, 50 and 100 ml/min/well respectively. CONCLUSIONS: QCMs within the Vitrocell® exposure module have demonstrated applicability at defining particle dose ranges at various experimental conditions. This tool will prove useful for users of the Vitrocell® system for dose-response determination and QC purposes.

Proteomic profiling of plasma in Huntington's disease reveals neuroinflammatory activation and biomarker candidates.

Huntington's disease (HD) causes widespread CNS changes and systemic abnormalities including endocrine and immune dysfunction. HD biomarkers are needed to power clinical trials of potential treatments. We used multiplatform proteomic profiling to reveal plasma changes with HD progression. Proteins of interest were evaluated using immunoblotting and ELISA in plasma from 2 populations, CSF and R6/2 mice. The identified proteins demonstrate neuroinflammation in HD and warrant further investigation as possible biomarkers.

Interleukin-1beta induces calcium transients and enhances basal and store operated calcium entry in human myometrial smooth muscle.

We have previously reported increased protein expression of sarcoplasmic reticulum calcium ATPase (SERCA) 2b in myometrium from women in labor at term, but the stimulus for this change is unknown. Proinflammatory cytokines have been implicated in the cascade of events leading to preterm and term labor, and we hypothesize that interleukin (IL)-1beta may induce changes in key calcium homeostatic mechanisms and, in turn, augment myometrial contractility before labor. The aim of the present study was to investigate the long-term effects of IL-1beta on SERCA 2b protein expression, calcium mobilization from intracellular stores, and store-operated calcium entry. Myometrial biopsies were obtained, with consent, from women undergoing elective cesarean section at term. Primary-cultured human myometrial smooth muscle (HMSM) cells were exposed to IL-1beta (10 ng/ml) for 24 h or to culture medium alone (control). Cells were subsequently used in Western blot studies or loaded with fura-2 to assess calcium dynamics using fluorescent digital imaging. The present study clearly demonstrated that IL-1beta significantly increased SERCA 2b protein expression in HMSM cells. Cyclopiazonic acid-induced calcium transients were also augmented, predominantly by activation of lanthanum-sensitive, store-operated calcium entry. HMSM cell excitability was enhanced, as evidenced by increased basal calcium entry and the initiation of spontaneous calcium transients in 37% of IL-1beta-treated cells. IL-1beta modulation of calcium mobilization may be an important mechanism in the cascade of events preparing the pregnant uterus for labor.