The Chemical Complexity of e-Cigarette Aerosols Compared With the Smoke From a Tobacco Burning Cigarette.

Background: As e-cigarette popularity has increased, there is growing evidence to suggest that while they are highly likely to be considerably less harmful than cigarettes, their use is not free of risk to the user. There is therefore an ongoing need to characterise the chemical composition of e-cigarette aerosols, as a starting point in characterising risks associated with their use. This study examined the chemical complexity of aerosols generated by an e-cigarette containing one unflavored and three flavored e-liquids. A combination of targeted and untargeted chemical analysis approaches was used to examine the number of compounds comprising the aerosol. Contributions of e-liquid flavors to aerosol complexity were investigated, and the sources of other aerosol constituents sought. Emissions of 98 aerosol toxicants were quantified and compared to those in smoke from a reference tobacco cigarette generated under two different smoking regimes. Results: Combined untargeted and targeted aerosol analyses identified between 94 and 139 compounds in the flavored aerosols, compared with an estimated 72-79 in the unflavored aerosol. This is significantly less complex (by 1-2 orders of magnitude) than the reported composition of cigarette smoke. Combining both types of analysis identified 5-12 compounds over and above those found by untargeted analysis alone. Gravimetrically, 89-99% of the e-cigarette aerosol composition was composed of glycerol, propylene glycol, water and nicotine, and around 3% comprised other, more minor, constituents. Comparable data for the Ky3R4F reference tobacco cigarette pointed to 58-76% of cigarette smoke "tar" being composed of minor constituents. Levels of the targeted toxicants in the e-cigarette aerosols were significantly lower than those in cigarette smoke, with 68.5->99% reductions under ISO 3308 puffing conditions and 88.4->99% reductions under ISO 20778 (intense) conditions; reductions against the WHO TobReg 9 priority list were around 99%. Conclusion: These analyses showed that the e-cigarette aerosols contain fewer compounds and at significantly lower concentrations than cigarette smoke. The chemical diversity of an e-cigarette aerosol is strongly impacted by the choice of e-liquid ingredients.

Mouth level smoke exposure using analysis of filters from smoked cigarettes: a study of eight countries.

The analysis of spent cigarette filters enables the estimation of the nicotine and tar (nicotine-free dry particulate matter) yields obtained by smokers in their everyday environment and has been shown to correlate well with biomarkers of exposure. Leading products across the range of ISO tar yields were selected from Australia, Brazil, Canada, Germany, Japan, New Zealand, South Africa and Switzerland. At least fifty demographically representative smokers were recruited per product. Subjects, ≥ 21 years of age and smoking ≥ 5 cigarettes per day, were asked to collect ≥ 15 filters from cigarettes they had smoked. The collected filters were analysed for nicotine and UV absorbance to enable the smokers' mouth level exposure to nicotine and tar to be estimated and a comparison of countries and tobacco blend styles to be made. Smoking history data were also collected. More than 80,000 filters were collected from 5703 smokers of 106 products from eight countries. Mean ± SD estimated nicotine exposures per cigarette and per day ranged from 0.93 ± 0.34 mg/cigarette (Brazil) to 1.77 ± 0.69 mg/cigarette (South Africa) and from 16.4 ± 11.1mg/day (Germany) to 31.5 ± 14.8 mg/day (South Africa), respectively. Male smokers obtained higher mean estimated tar and nicotine exposures than female smokers. These gender differences were statistically significant for six countries. Significant correlations were found between estimated nicotine exposure and ISO nicotine yield, and between estimated tar exposure and ISO tar yield (p<0.001).

Post-puff respiration measures on smokers of different tar yield cigarettes.

The purpose of this study was to determine the effect of different tar yield cigarette brands on the post-puff inhalation/exhalation depth and duration for established smokers of the brands. The study was conducted with 74 established smokers of 1-17 mg Federal Trade Commission (FTC) tar products. The subjects were participating in a five-day inpatient clinical biomarker study during which time they were allowed to smoke their own brand of cigarette whenever they wished. On two separate days, the subjects' breathing pattern was measured using respiratory inductive plethysmography while they smoked one cigarette. This enabled the measurement of the post-puff inhalation volume, exhalation volume, inhalation duration, and exhalation duration for each subject after each puff on two of their own brand of cigarettes. The subjects were grouped according to the FTC tar yield of their product: 1-3 mg; 4-6 mg; 7-13 mg; 14 + mg. The post-puff inhalation volume for the 4-6 mg group was significantly lower than both the 7-13 mg and 14+ mg groups, and the 4-6 mg group exhalation volume was significantly lower than the 14+ mg group (p < 0.05). No other differences were found at the 95% confidence level. When volumes were normalized to resting tidal volume (tidal ratio), there were no differences between the groups for any of the respiratory measures. No significant slope was found for correlations with FTC tar yield for inhalation volume (p = 0.11, mean = 833 mL, R = 0.19), inhalation tidal ratio (p = 0.93, mean = 1.73, R = -0.01) or lung exposure time (p = 0.92, mean = 4.1 s, R = -0.01).

A comparison of nicotine dose estimates in smokers between filter analysis, salivary cotinine, and urinary excretion of nicotine metabolites.

RATIONALE: Nicotine uptake during smoking was estimated by either analyzing the metabolites of nicotine in various body fluids or by analyzing filters from smoked cigarettes. However, no comparison of the filter analysis method with body fluid analysis methods has been published. OBJECTIVES: Correlate nicotine uptake estimates between filter analysis, salivary cotinine, and urinary excretion of selected nicotine metabolites to determine the suitability of these methods in estimating nicotine absorption in smokers of filtered cigarettes. MATERIALS AND METHODS: A 5-day clinical study was conducted with 74 smokers who smoked 1-19 mg Federal Trade Commission tar cigarettes, using their own brands ad libitum. Filters were analyzed to estimate the daily mouth exposure of nicotine. Twenty-four-hour urine samples were collected and analyzed for nicotine, cotinine, and 3'-hydroxycotinine plus their glucuronide conjugates. Saliva samples were collected daily for cotinine analysis. RESULTS: Each method correlated significantly (p < 0.01) with the other two. The best correlation was between the mouth exposure of nicotine, as estimated by filter analysis, and urinary nicotine plus metabolites. Multiple regression analysis implies that saliva cotinine and urinary output are dependent on nicotine mouth exposure for multiple days. Creatinine normalization of the urinary metabolites degrades the correlation with mouth exposure. CONCLUSIONS: The filter analysis method was shown to correlate with more traditional methods of estimating nicotine uptake. However, because filter analysis is less complicated and intrusive, subjects can collect samples easily and unsupervised. This should enable improvements in study compliance and future study designs.

Identification of Src phosphorylation sites in the catenin p120ctn.

p120-catenin (p120(ctn)) interacts with the cytoplasmic tail of cadherins and is thought to regulate cadherin clustering during formation of adherens junctions. Several observations suggest that p120 can both positively and negatively regulate cadherin adhesiveness depending on signals that so far remain unidentified. Although p120 tyrosine phosphorylation is a leading candidate, the role of this modification in normal and Src-transformed cells remains unknown. Here, as a first step toward pinpointing this role, we have employed two-dimensional tryptic mapping to directly identify the major sites of Src-induced p120 phosphorylation. Eight sites were identified by direct mutation of candidate tyrosines to phenylalanine and elimination of the accompanying spots on the two-dimensional maps. Identical sites were observed in vitro and in vivo, strongly suggesting that the physiologically important sites have been correctly identified. Changing all of these sites to phenylalanine resulted in a p120 mutant, p120-8F, that could not be efficiently phosphorylated by Src and failed to interact with SHP-1, a tyrosine phosphatase shown previously to interact selectively with tyrosine-phosphorylated p120 in cells stimulated with epidermal growth factor. Using selected tyrosine to phenylalanine p120 mutants as dominant negative reagents, it may now be possible to selectively block events postulated to be dependent on p120 tyrosine phosphorylation.

Inhibition of RhoA by p120 catenin.

RhoA organizes actin stress fibres and is necessary for cell transformation by oncogenes such as src and ras. Moreover, RhoA is implicated in cadherin clustering during the formation of adherens junctions. The catenin p120 has also been implicated in cadherin clustering through an unknown mechanism. Here we show that p120 selectively inhibits RhoA activity in vitro and in vivo. RhoA inhibition and the interaction of p120 with cadherins are mutually exclusive, suggesting a mechanism for regulating the recruitment and exchange of RhoA at nascent cell-cell contacts. By affecting RhoA activation, p120 could modulate cadherin functions, including suppression of invasion, neurite extension and junction formation.

ARVCF localizes to the nucleus and adherens junction and is mutually exclusive with p120(ctn) in E-cadherin complexes.

ARVCF is a novel Armadillo repeat domain protein that is closely related to the catenin p120(ctn). Using new ARVCF monoclonal antibodies, we have found that ARVCF associates with E-cadherin and competes with p120 for interaction with the E-cadherin juxtamembrane domain. ARVCF also localized to the nucleus in some cell types, however, and was significantly more nucleophilic than p120. Surprisingly, despite apparently ubiquitous expression, ARVCF was at least tenfold less abundant than p120 in a wide variety of cell types, and was difficult to detect by immunofluorescence unless overexpressed. Consequently, it is not likely to be abundant enough in adult tissues to functionally compete with p120. ARVCF also completely lacked the ability to induce the cell-branching phenotype associated with overexpression of p120. Expression of ARVCF/p120 chimeras confirmed previous results indicating that the branching activity of p120 maps to its Armadillo repeat domain. Surprisingly, the preferential localization of ARVCF to the nucleus required sequences in the amino-terminal end of ARVCF, suggesting that the sequences directing nuclear translocation of ARVCF are distinct from the predicted bipartite nuclear localization signal located between repeats 6 and 7. The dual localization of ARVCF to junctions and to nuclei suggests activities in different cellular compartments, as is the case for several other Armadillo repeat proteins including beta-catenin, p120 and the plakophilins.

Production and characterization of monoclonal antibodies to ARVCF.

We have generated the first monoclonal antibodies (MAbs) to Armadillo repeat gene deleted in velo-cardiofacial syndrome (ARVCF), a recently identified Armadillo repeat-containing protein closely related to the catenin p120ctn. Six ARVCF-specific MAbs were characterized for isotype, species cross-reactivity, and utility in assays including immunofluorescence, immunoprecipitation, and Western blotting. All six antibodies were isotyped as IgG1 and several cross-reacted with ARVCF from a variety of species including human, rat, dog, and monkey, but not mouse. Importantly, none of the ARVCF MAbs cross-reacted with p120ctn, despite the high homology between these proteins. MAbs 3B2 and 4B1 were consistently the best in all applications and will provide valuable tools for further study of the role of ARVCF in cells.

Production and characterization of monoclonal antibodies to the catenin p120ctn.

This report describes the generation and characterization of a panel of monoclonal antibodies (MAb) to the catenin p120ctn. p120ctn (formerly p120cas) is a cadherin-binding protein with structural similarity to the classical catenins beta-catenin and plakoglobin. It was originally identified as a prominent Src substrate and subsequently as a substrate for the Platelet Derived Growth Factor (PDGF), Epidermal Growth Factor (EGF), and Colony Stimulating Factor-1 (CSF-1) receptor tyrosine kinases. To facilitate further study of p120 function, we have generated novel MAbs to both the N- and C-terminal ends of p120 and compared them to previously described antibodies to these regions.

Long-term effects of switching to cigarettes with lower tar and nicotine yields.

On switching to cigarettes with lower tar and nicotine yields, most individuals smoke more intensively, but it is not clear if this effect persists over a long period. Smoking behaviour was monitored in 10 male and 18 female volunteers at five monthly visits, smoking commercially available cigarettes (tar yield greater than 10 mg), then for six more visits at 6-week intervals after switching (mean reduction of 5.9 mg tar and 0.45 mg nicotine). Puffing behaviour was monitored with a flow sensing holder, and measurements were made before and after smoking of plasma cotinine, carboxyhaemglobin and alveolar carbon monoxide. After switching, cotinine levels only fell 40% of that predicted from the fall in nicotine yields, and there were no systematic trends for the rest of the study. Puff volumes rose (reflecting perhaps the reduced draw resistance of the lower yield cigarettes), and remained higher thereafter. The number of puffs per cigarette appeared to rise on switching, but then decreased again. In conclusion, most effects of switching to lower yield cigarettes appeared to persist for at least 36 weeks, suggesting that the strategy of reducing exposure to cigarette smoke by lowering tar and nicotine yields may be of limited value.

The Kensey catheter: preliminary results with a transluminal atherectomy tool.

The Kensey dynamic angioplasty instrument is an atherectomy device approved by the Food and Drug Administration that uses a rotating cam tip housed within a flexible polyurethane catheter to recanalize obstructed and stenotic arteries. Twenty patients with significant femoral arteriosclerotic occlusive disease underwent attempted transluminal endarterectomy of 23 extremities with the Kensey catheter. Significant improvements of superficial femoral artery luminal diameter was achieved in 10 of 13 patients with stenosis and passage of the spinning catheter tip at 60,000 to 90,000 rpm through areas of complete occlusion was successful in 4 of 10 cases. Balloon dilatation was used as an adjunct to increase the diameter of the superficial femoral artery lumen in 11 of 14 successful cases. This preliminary report provides technical data and short-term follow-up of this new innovative vascular tool.

Is alveolar carbon monoxide an unreliable index of carboxyhaemoglobin changes during smoking in man?

1. We measured alveolar carbon monoxide (CO) after a 20 s breath-holding period and carboxyhaemoglobin both before and after smoking a cigarette on 500 occasions (101 individuals). The two measurements were closely correlated but there was a marked difference in the change or 'boost' after smoking one cigarette. The mean relative boosts ([post value--pre value]/[pre+post]/2) for alveolar CO and carboxyhaemoglobin were 7.7% and 20.3%, while negative boosts (fall rather than the expected rise) were seen in 103 of 500 and three of 500 occasions respectively. In 140 studies a third alveolar CO reading taken 5 min later was slightly larger, but the difference was insignificant. 2. In seven subjects where the carboxyhaemoglobin level was raised by breathing a 2% CO gas mixture, the alveolar CO and carboxyhaemoglobin boosts were similar (71.7% and 75.2% respectively), and they fell sharply subsequently rather than increasing further as occurred after smoking. 3. We conclude that alveolar CO measurements give a useful estimate of carboxyhaemoglobin level if the subject has not smoked for at least half an hour but that measurements of alveolar CO boost are useless since the act of smoking interferes with alveolar sampling. We postulate that cigarette smoking induces a transient change in pulmonary gas exchange.

Vocal cord paralysis following I-131 ablation of a postthyroidectomy remnant.

Vocal cord paralysis has been reported following I-131 therapy of thyrotoxicosis and following ablation of the whole thyroid. However, this rare complication has not previously been described following I-131 ablation of a postthyroidectomy remnant. We report a patient who required tracheostomy for bilateral vocal cord paralysis following I-131 ablation after near-total thyroidectomy for papillary thyroid carcinoma.