Impact of chronic smoking on traumatic brain microvascular injury: An in vitro study.

Traumatic brain injury (TBI) is a major reason of cerebrovascular and neurological damage. Premorbid conditions such as tobacco smoking (TS) can worsen post-TBI injuries by promoting vascular endothelial impairments. Indeed, TS-induced oxidative stress (OS) and inflammation can hamper the blood-brain barrier (BBB) endothelium. This study evaluated the subsequence of chronic TS exposure on BBB endothelial cells in an established in vitro model of traumatic cell injury. Experiments were conducted on confluent TS-exposed mouse brain microvascular endothelial cells (mBMEC-P5) following scratch injury. The expression of BBB integrity-associated tight junction (TJ) proteins was assessed by immunofluorescence imaging (IF), Western blotting (WB) and quantitative RT-PCR. We evaluated reactive oxygen species (ROS) generation, the nuclear factor 2-related (Nrf2) with its downstream effectors and several inflammatory markers. Thrombomodulin expression was used to assess the endothelial haemostatic response to injury and TS exposure. Our results show that TS significantly decreased Nrf2, thrombomodulin and TJ expression in the BBB endothelium injury models while increased OS and inflammation compared to parallel TS-free cultures. These data suggest that chronic TS exposure exacerbates traumatic endothelial injury and abrogates the protective antioxidative cell responses. The downstream effect was a more significant decline of BBB endothelial viability, which could aggravate subsequent neurological impairments.

Aqueous cigarette tar extracts disrupt corticogenesis from human embryonic stem cells in vitro.

BACKGROUND: Cigarette butts are the most common form of litter in the world, and approximately 4.5 trillion smoked cigarettes are discarded every year worldwide. Cigarette butts contain over 4000 chemicals, many of which are known to have neurotoxic effects. Stem cell neuronal differentiation provides an excellent cellular model with which to examine the impact of aqueous cigarette tar extracts (ACTEs) on neurodevelopment. METHODS: We have developed a neurosphere-based stem cell neuronal differentiation protocol that can recapitulate corticogenesis and produce cell types that are similar to upper and lower layer cortical projection neurons found in the germinal zone of the developing human cortex. In this study, ACTEs were generated from smoked cigarette butts and then applied at different concentrations to neuronal progenitors and cortical neurons derived from human embryonic stem cells. RESULTS: ACTEs reduced the expression of the cortical neuronal progenitor markers pax6, tbr2, and neuroD and decreased the number of cortical layer neurons (tbr1, satb2, foxp2, and brn2) after exposure to as low as 1.87% of the extract from one smoked cigarette butt. Furthermore, our results showed that ACTEs increased reactive oxygen species (ROS) production in cortical neurons, which caused a substantial loss of the synaptic proteins PSD95, synaptophysin, vesicular glutamate transporter1 (vGlut1), and the extracellular matrix molecule reelin; all of those molecules are important for the maintenance of cortical neuron identity and activity. CONCLUSION: ACTEs from smoked cigarettes have significant effects on cortical neuron development and neurodegeneration. The stem cell neuronal differentiation model holds great promise as a potentially powerful tool for the assessment of ACTEs on neurotoxicity.

The activation of M3 mAChR in airway epithelial cells promotes IL-8 and TGF-ß1 secretion and airway smooth muscle cell migration.

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) have been identified in airway epithelium, and epithelium-derived chemokines can initiate the migration of airway smooth muscle (ASM) cells. However, the mAChRs that are expressed in airway epithelium and the mechanism underlying the regulation of ASM cell migration are not clear. The aim of this study was to test whether the effects of the epithelium-derived chemokines on ASM cell migration could be modulated by mAChRs. METHOD: Human epithelial cells (A549 cells) were stimulated with cigarette smoke extract (CSE) or the mAChRs agonist carbachol. IL-8 and TGF-ß1 production were measured by ELISA, and human ASM cell migration was measured using the transwell migration assay and scratch assay. The mRNA levels of the mAChRs subtypes and the acetylcholine concentrations were measured using RT-PCR and LC-MS/MS, respectively. RESULTS: ASM cell migration toward CSE-stimulated A549 cells was markedly reduced by Ac-RRWWCR-NH2 (IL-8 inhibitor) and SB431542 (TGF-ß1 inhibitor). CSE-induced ASM cell migration was also suppressed by the mAChRs antagonist tiotropium. Interestingly, carbachol-stimulated A549 cells also induced ASM cell migration; this migration event was suppressed by tiotropium, Ac-RRWWCR-NH2 and SB431542. In addition, the effects of CSE on ASM cell migration were significantly and cooperatively enhanced by carbachol compared to CSE alone. Carbachol-induced ASM cell migration was reduced by selective inhibitors of PI3K/Akt (LY294002) and p38 (SB203580), suggesting that it occurred through p38 and Akt phosphorylation, which was inhibited by the M3 mAChR antagonist 4-DAMP. CONCLUSIONS: These findings indicate that M3 mAChR may be important therapeutic target for obstructive airway diseases, as it regulates the effects of the epithelial-derived chemokines on ASM cell migration, which results in lung remodeling.

Crosstalk Between Co-cultured A549 Cells and THP1 Cells Exposed to Cigarette Smoke.

Cigarette smoke (CS) is considered as a major etiological factor in the pathogenesis of chronic obstructive pulmonary disease. In this study we used A549 cells and THP-1 cells grown for 24 h in monoculture or in co-culture in CS-conditioned media and changes in their proliferation, viability, acetylated histone H3 levels and expression of extracellular antigens CD14, HLA-DR, CD11a, and CD11b were assessed. CS was highly toxic to A549 cells but not to THP1 cells. In A549 cells, oxidative stress reached the highest values after 1 h of CS exposure and then decreased. In THP1 cells oxidative stress was lower and increased progressively with time. CS decreased proliferation of A549 and THP1 cells by about 80% and 21%, respectively. CS did not alter acetylated histone H3 levels in A549 cells, while in THP1 cells the levels were reduced by about 35%. CS significantly increased expression of CD14, HLA-DR, CD11a, and CD11b in THP1 cells. In co-culture, naïve or CS-pretreated THP1 cells significantly protected A549 cells against CS toxicity but had higher death rates. These results show that epithelial cells are more fragile to CS than monocytes and that CS-activated monocytes may protect epithelial cells against CS-induced cytotoxicity.

The assessment of sewage sludge gasification by-products toxicity by ecotoxicologial test.

The process of gasification of sewage sludge generates by-products, which may be contaminated with toxic and hazardous substances, both organic and inorganic. It is therefore important to assess the environmental risk associated with this type of waste. The feasibility of using an ecotoxicological tests for this purpose was determined in the presented study. The applied tests contained indicator organisms belonging to various biological groups (bacteria, crustaceans, plants). The subject of the study were solid (ash, char) and liquid (tar) by-products generated during gasification (in a fixed bed reactor) of dried sewage sludge from various wastewater treatment systems. The tested samples were classified based on their toxic effect. The sensitivity of the indicator organisms to the tested material was determined. In-house procedures for the preparation for toxicity analysis of both sewage sludge and by-products generated during the gasification were presented. The scope of work also included the determination of the effect of selected process parameters (temperature, amount of gasifying agent) on the toxicity of gasification by-products depending on the sewage sludge source. It was shown that both the type of sewage sludge and the parameters of the gasification process affects the toxicity of the by-products of gasification. However, the results of toxicity studies also depend on the type of ecotoxicological test used, which is associated with a different sensitivity of the indicator organisms. Nevertheless, it may be concluded that the by-products formed during the gasification of the low toxicity sewage sludge can be regarded as non-toxic or low toxic. However, the results analysis of the gasification of the toxic sludge were not conclusive, which leads to further research needs in this area.

Aggravation of ovalbumin-induced murine asthma by co-exposure to desert-dust and organic chemicals: an animal model study.

BACKGROUND: The organic chemicals present in Asian sand dust (ASD) might contribute to the aggravation of lung eosinophila. Therefore, the aggravating effects of the Tar fraction from ASD on ovalbumin (OVA)-induced lung eosinophilia were investigated. METHODS: The Tar fraction was extracted from ASD collected from the atmosphere in Fukuoka, Japan. ASD collected from the Gobi desert was heated at 360°C to inactivate toxic organic substances (H-ASD). ICR mice were instilled intratracheally with 12 different test samples prepared with Tar (1 µg and 5 µg), H-ASD, and OVA in a normal saline solution containing 0.02% Tween 80. The lung pathology, cytological profiles in the bronchoalveolar lavage fluid (BALF), inflammatory cytokines/chemokines in BALF and OVA-specific immunoglobulin in serum were investigated. RESULTS: Several kinds of polycyclic aromatic hydrocarbons (PAHs) were detected in the Tar sample. H-ASD + Tar 5 µg induced slight neutrophilic lung inflammation. In the presence of OVA, Tar 5 µg increased the level of eosinophils slightly and induced trace levels of Th2 cytokines IL-5 and IL-13 in BALF. Also mild to moderate goblet cell proliferation and mild infiltration of eosinophils in the submucosa of airway were observed. These pathological changes caused by H-ASD + OVA were relatively small. However, in the presence of OVA and H-ASD, Tar, at as low a level as 1 µg, induced severe eosinophil infiltration and proliferation of goblet cells in the airways and significantly increased Th2 cytokines IL-5 and IL-13 in BALF. The mixture showed an adjuvant effect on OVA-specific IgG1 production. CONCLUSIONS: These results indicate that H-ASD with even low levels of Tar exacerbates OVA-induced lung eosinophilia via increases of Th2-mediated cytokines. These results suggest that ASD-bound PAHs might contribute to the aggravation of lung eosinophila.

Reduced exposure evaluation of an Electrically Heated Cigarette Smoking System. Part 1: Non-clinical and clinical insights.

The following series of papers presents an extensive assessment of the Electrically Heated Cigarette Smoking System EHCSS series-K cigarette vs. conventional lit-end cigarettes (CC) as an example for an extended testing strategy for evaluation of reduced exposure. The EHCSS produces smoke through electrical heating of tobacco. The EHCSS series-K heater was designed for exclusive use with EHCSS cigarettes, and cannot be used to smoke (CC). Compared to the University of Kentucky Reference Research cigarette 2R4F and a series of commercial CC, mainstream cigarette smoke of both the non-menthol and menthol-flavored EHCSS cigarettes showed a reduced delivery of a series of selected harmful and potentially harmful constituents (HPHC), mutagenic activity determined using the Salmonella typhimurium Reverse Mutation (Ames) assay, and cytotoxicity in the Neutral Red Uptake Assay. Clinical evaluations confirmed reduced exposure to HPHC and excretion of mutagenic material under controlled clinical conditions. Reductions in HPHC exposure were confirmed in a real-world ambulatory clinical study. Potential biomarkers of cardiovascular risk were also reduced under real-world ambulatory conditions. A modeling approach, 'nicotine bridging', was developed based on the determination of nicotine exposure in clinical evaluations which indicated that exposure to HPHC for which biomarkers of exposure do not exist would also be reduced.

Reduced exposure evaluation of an Electrically Heated Cigarette Smoking System. Part 8: Nicotine bridging--estimating smoke constituent exposure by their relationships to both nicotine levels in mainstream cigarette smoke and in smokers.

A modeling approach termed 'nicotine bridging' is presented to estimate exposure to mainstream smoke constituents. The method is based on: (1) determination of harmful and potentially harmful constituents (HPHC) and in vitro toxicity parameter-to-nicotine regressions obtained using multiple machine-smoking protocols, (2) nicotine uptake distributions determined from 24-h excretion of nicotine metabolites in a clinical study, and (3) modeled HPHC uptake distributions using steps 1 and 2. An example of 'nicotine bridging' is provided, using a subset of the data reported in Part 2 of this supplement (Zenzen et al., 2012) for two conventional lit-end cigarettes (CC) and the Electrically Heated Cigarette Smoking System (EHCSS) series-K6 cigarette. The bridging method provides justified extrapolations of HPHC exposure distributions that cannot be obtained for smoke constituents due to the lack of specific biomarkers of exposure to cigarette smoke constituents in clinical evaluations. Using this modeling approach, exposure reduction is evident when the HPHC exposure distribution curves between the MRTP and the CC users are substantially separated with little or no overlap between the distribution curves.

Reduced exposure evaluation of an Electrically Heated Cigarette Smoking System. Part 2: Smoke chemistry and in vitro toxicological evaluation using smoking regimens reflecting human puffing behavior.

Chemical analysis of up to 49 harmful and potentially harmful constituents (HPHC) in mainstream smoke, in vitro cytotoxicity of the particulate and gas/vapor phase of mainstream smoke determined in the Neutral Red Uptake assay, and in vitro bacterial mutagenicity of the particulate phase determined in the Salmonella typhimurium Reverse Mutation (Ames) assay are reported for three Electrically Heated Cigarette Smoking System (EHCSS) series-K cigarettes, the University of Kentucky Reference Cigarette 2R4F, and a number of comparator commercial conventional lit-end cigarettes (CC) under ISO machine-smoking conditions and a total of 25 additional smoking regimens reflecting 'human puffing behavior' (HPB). The smoking machines were set to deliver nicotine yields for the EHCSS and comparator CC derived from the 10th percentile to the 90th percentile of nicotine uptake distributions in smokers determined in two clinical studies. Duplication of the smoking intensity 'per cigarette' on a smoking machine may provide an insight into product performance that is directly relevant to obtaining scientific evidence for reduced exposure substantiation based on mainstream cigarette smoke HPHC-to-nicotine regressions. The reported data support an overall evaluation of reduced exposure to HPHC and biological activity.

Bronchial morphometry in smokers: comparison with healthy subjects by using 3D CT.

The assessment of airway dimensions in patients with airway disease by using computed tomography (CT) has been limited by the obliquity of bronchi, the ability to identify the bronchial generation, and the limited number of bronchial measurements. The aims of the present study were (i) to analyze cross-sectional bronchial dimensions after automatic orthogonal reconstruction of all visible bronchi on CT images, and (ii) to compare bronchial morphometry between smokers and nonsmokers. CT and pulmonary function tests were performed in 18 males separated into two groups: 9 nonsmokers and 9 smokers. Bronchial wall area (WA) and lumen area (LA) were assessed using dedicated 3D software able to provide accurate cross-sectional measurements of all visible bronchi on CT. WA/LA and WA/(WA+LA) ratios were computed and all parameters were compared between both groups. Smokers demonstrated greater WA, smaller LA, and consequently greater LA/WA and LA/(WA+LA) ratios than nonsmokers. These differences occurred downward starting at the fourth bronchial generation. 3D quantitative CT method is able to demonstrate significant changes in bronchial morphometry related to tobacco consumption.

gammaH2AX: A potential DNA damage response biomarker for assessing toxicological risk of tobacco products.

Differentiation among American cigarettes relies primarily on the use of proprietary tobacco blends, menthol, tobacco substitutes, paper porosity, paper additives, and filter ventilation. These characteristics substantially alter per cigarette yields of tar and nicotine in standardized protocols promulgated by government agencies. However, due to compensatory alterations in smoking behavior to sustain a preferred nicotine dose (e.g., by increasing puff frequency, inhaling more deeply, smoking more cigarettes per day, or blocking filter ventilation holes), smokers actually inhale similar amounts of tar and nicotine regardless of any cigarette variable, supporting epidemiological evidence that all brands have comparable disease risk. Consequently, it would be advantageous to develop assays that realistically compare cigarette smoke (CS)-induced genotoxicity regardless of differences in cigarette construction or smoking behavior. One significant indicator of potentially carcinogenic DNA damage is double strand breaks (DSBs), which can be monitored by measuring Ser 139 phosphorylation on histone H2AX. Previously we showed that phosphorylation of H2AX (defined as gammaH2AX) in exposed lung cells is proportional to CS dose. Thus, we proposed that gammaH2AX may be a viable biomarker for evaluating genotoxic risk of cigarettes in relation to actual nicotine/tar delivery. Here we tested this hypothesis by measuring gammaH2AX levels in A549 human lung cells exposed to CS from a range of commercial cigarettes using various smoking regimens. Results show that gammaH2AX induction, a critical event of the mammalian DNA damage response, provides an assessment of CS-induced DNA damage independent of smoking topography or cigarette type. We conclude that gammaH2AX induction shows promise as a genotoxic bioassay offering specific advantages over the traditional assays for the evaluation of conventional and nonconventional tobacco products.

[Carcinogenic effect of tobacco smoke]. / Rakotwórcze dzialanie dymu tytoniowego.

Both epidemiological and experimental studies provide evidence of the dose-effect relationship between the number of cigarettes smoked and lung cancer risk, exposure to tar or tobacco smoke and skin cancers or squamous cell carcinoma of the trachea and lung. Polycyclic aromatic hydrocarbons (PAHs) and volatile N-nitrosamines, and also tobacco specific N-nitrosamines are considered to be the major carcinogens in tobacco smoke. To exert carcinogenic effect these compounds require previous metabolic activation by biotransformation enzymes. Individual susceptibility to chemical carcinogens is genotype and phenotype dependent. Machine-measured yields of tar, nicotine, carbon monoxide, benzo[a]pyrene and N-nitrosonornicotine in cigarette smoke are significantly lower than actual intake by smokers. The following features have significant influence on the tobacco smoke composition, cancer risk and other disease risks relative to cigarette smoking: tobacco type and its modifications and also nitrate content in tobacco. Tobacco additives, including ammonia releasing substances, do not contribute to cigarette smoke composition and its toxicity. Filters, paper porosity, cigarette length and circumference as well as the number of tobacco cuts per inch (whether it is coarse-cut or fine-cut tobacco) are of primary significance for the chemical composition of cigarette smoke and health risk.

Exposure level to cigarette tar or nicotine is associated with leukocyte DNA damage in male Japanese smokers.

We investigated the number of cigarettes smoked daily, years of smoking, cigarette pack-years, levels of daily exposure to cigarette tar (LECT, mg/day) or nicotine (LECN, mg/day) in 53 male Japanese smokers using a questionnaire and measured each participant's baseline leukocyte DNA damage using the alkaline comet assay. The results showed that the baseline value of peripheral leukocyte DNA strand breaks was significantly associated with LECT (P < 0.05), LECN (P < 0.05), years of smoking or cigarette pack-years (P < 0.05) but not with the number of cigarettes smoked per day. Stepwise multiple regression analyses of factors including age, occupation, years of employment, alcohol drinking behaviour, physical activity, nutritional balance and cigarette smoking parameters showed that LECT was a positively significant predictor (Partial r = 0.0005, P < 0.05) of the comet tail moment. In consideration of the high correlation between LECT and LECN (Y(tar) = 12.53 X(nicotine) -7.23, r = 0.995, P < 0.0001), these results suggest that levels of exposure to cigarette tar or nicotine (mg/day) would be a sensitive parameter in appreciation of genotoxicity of cigarette smoking in these male Japanese smokers.

On the skin sensitisation potential of rosin and oxidised rosin.

In the EU rosin is classified as a skin sensitiser, apparently on the basis of its oxidation to sensitising agents. Rosin (gum, tall oil or wood) is not a skin sensitiser when examined in the guinea pig maximisation test (GPMT). Oxidised rosins are sensitisers in the GPMT. Oxidised gum rosin was further tested in the mouse local lymph node assay (LLNA) and the Buehler test, but is not a sensitiser in either of these tests. Further, the outcome of the LLNA can be used to assess the potency of oxidised rosin as an inducing agent in humans, and oxidised rosin is, at most, a weak sensitiser in this test. Thus, oxidised rosin is not a potent inducing agent for skin sensitisation unless the dermal barrier is bypassed and/or there is deliberate use of Freund's Complete Adjuvant to induce greater susceptibility. The material used for human patch testing ('colophony') is in oxidised form. A re-examination of epidemiological studies suggests that patients in dermatological clinics show higher response rates than do the general population or those occupationally exposed to presumably oxidised rosin. Thus, the differences seen in susceptibility in the regulatory tests may be reflected in the human population. These results are discussed in terms of possible testing and classification strategies for dealing with existing chemicals, with particular reference to the new European Union legislation.

Safety assessment of diammonium phosphate and urea used in the manufacture of cigarettes.

A tiered testing strategy has been employed to evaluate the potential for new ingredients, tobacco processes, and technological developments to alter the mainstream smoke or biological activity that results from burning cigarette tobacco. The foundation of this evaluation strategy is comparative testing, typically including chemical and biological assessments. In the manufacture of cigarettes, diammonium phosphate (DAP) and urea have been historically used as ingredients added to tobacco, to reconstituted tobacco sheet, and to other processed tobaccos. As part of ongoing stewardship efforts, a toxicological assessment of cigarettes with and without DAP and urea was conducted. Chemical and biological analyses were conducted for test cigarettes added 0.5% DAP and 0.2% urea in the final blend and also for those added 1.0% DAP and 0.41% urea in the final blend compared to reference cigarettes without added DAP or urea. Principal components of this evaluation included a determination of selected mainstream smoke constituent yields, an Ames assay in Salmonella typhimurium strains TA98 and TA100, a sister chromatid exchange assay in Chinese hamster ovary cells, a 13-week inhalation study of mainstream cigarette smoke in Sprague-Dawley rats, and a 30-week dermal tumor-promotion evaluation of mainstream cigarette smoke condensate in SENCAR mice. Comparative evaluations demonstrated that the addition of DAP and urea to cigarettes at up to 1% and 0.41%, respectively, does not alter the biological activity compared to reference cigarettes without DAP or urea.

Cigarette tar phenols impede T cell cycle progression by inhibiting cyclin-dependent kinases.

Cigarette smoking causes profound suppression of pulmonary T cell responses, which is associated with increased susceptibility to respiratory tract infections and decreased tumor surveillance. We previously demonstrated that the phenolic compounds in cigarette tar inhibit blastogenesis and interfere with human T cell cycle progression. To identify the mechanism by which cell cycle arrest occurs, we examined the effects of these compounds on cyclin-dependent kinases (Cdk) that control the G0/G1 transition. We found that hydroquinone inhibited induction of Cdk4 and Cdk6 kinase activities by >80%, while catechol and phenol were markedly less potent. HQ did not affect mitogenic induction of the Cdk6 protein, but inhibited expression of cyclin D3 by >90% resulting in a dramatic reduction in proper Cdk6/Cyclin D3 complex formation.

Cytotoxicity of cigarette smoke condensate is not due to DNA double strand breaks: Comparative studies using radiosensitive mutant and wild-type CHO cells.

PURPOSE: To determine whether cigarette smoke condensate (CSC) without metabolic activation induces direct DNA double strand breaks (DSB) in the G1 phase of various radiosensitive mutants of CHO cells and whether these breaks display collateral hypersensitivity to CSC with respect to cell killing. MATERIALS & METHODS: We treated the G1-phase cultures of wild-type and DNA repair deficient mutants of CHO cells with various concentrations of CSC and examined the cell survival by colony formation assay and the induction of DNA double strand breaks by constant field gel electrophoresis as well as the phophorylated histone H2-A variant X (gamma-H2AX) assay. RESULTS: Gel analysis and gamma-H2AX focus assay showed significantly fewer, but still detectable levels of DSB per cell after CSC treatment compared to ionizing radiation (IR) exposures, even when equitoxic radiation exposures were delivered at a low dose rate over the same 8-hour exposure used for CSC treatments. None of the three non-homologous end joining (NHEJ) deficient mutants were remarkably hypersensitive to CSC compared to wild-type cells. In contrast, UV-1 cells that are hypersensitive to several base damage and cross-linking agents showed a higher sensitivity to CSC compared to the other CHO cell lines. CONCLUSIONS: DNA DSB produced directly by CSC are not principally responsible for its cytotoxicity. Further, the present study does not rule out the possibility that some of these lesions may secondarily result in DSB, such as may occur during impeded DNA replication and whose repair may require systems other than NHEJ.

Cigarette smoke condensate increases cathepsin-mediated invasiveness of oral carcinoma cells.

Cigarette smoke, which contains several carcinogens known to initiate and promote tumorigenesis and metastasis, is the major cause of oral cancer. Lysosomal cathepsin proteases play important roles in tumor progression, invasion and metastasis. In the present work we investigated the effects of cigarette smoke condensate (CSC) on cathepsin (B, D and L) expression and protease-mediated invasiveness in human oral squamous cell carcinoma (OSCC) cells. Our results show that treatment of OSCC cells (686Tu and 101A) with CSC activated cathepsins B, D and L in a dose-dependent manner. Both expression and activity of these cathepsins were up-regulated in CSC-exposed versus non-exposed cells. Although cathepsin L had the lowest basal level, it had the highest induction in exposed cells compared to cathepsins B and D. Suppression of CSC-induced cathepsin B and L activities by specific chemical inhibitors decreased the invasion process, suggesting that these proteases are involved in the invasion process. Overall, our results indicate that CSC activates cathepsin B and L proteolytic activity and enhances invasiveness in OSCC cells, a response that may play a role in CSC-mediated tumor progression and metastasis dissemination.

Dependence of tar, nicotine and carbon monoxide yields on physical parameters: implications for exposure, emissions control and monitoring.

OBJECTIVE: To estimate the extent to which tar, nicotine and carbon monoxide (TNCO) yields are dependent on cigarette design features such as burn rate, filter ventilation and paper porosity, and to consider the implications for human exposure and the regulation of TNCO emissions. A related aim is to determine whether accurate prediction of TNCO yields is possible using only simple physical parameters. DESIGN AND METHODS: Datasets that include quantitative design parameters as well as measurements of TNCO yields collected under standard conditions with vents unblocked (International Organization for Standardization) and under intense conditions with vents fully blocked (Health Canada) were compiled from the literature (primarily US and UK brands). Forward stepwise multiple regression analysis is used to assess the relative importance of each design feature in explaining variability in the observed emissions. Using randomly split data subsets, multiple linear regression is used to model the dependence of TNCO yields on design features in the training subset and validated against the test subset. Tar and carbon monoxide correlate with many of the particulate- and volatile-phase toxins in smoke, and brand values normalised to nicotine yield are used as surrogate measures of exposure within the bounds defined by non-intense and intense smoking protocols. RESULTS AND CONCLUSIONS: Filter ventilation is the dominant control on measured TNCO emissions, but other factors including burn rate, amount of tobacco and paper porosity also contribute. Yields are predictable with reasonable accuracy and precision using only measured physical parameters. Surrogate exposure indicators suggest that filter ventilation does not lead to any reduction in exposure and that highly ventilated (low-yield) brands may actually increase exposure to the more volatile toxins.