Particulate Phase Of Cigarette Smoke Research Articles

Comparison of Mainstream Smoke Composition from CR20 Resin Filter and Empty-Cavity Filter Cigarettes by Headspace SPME Coupled with GC×GC TOFMS and Chemometric Analysis

Summary A previously established method based on headspace solidphase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry (TOFMS) has been used to evaluate and compare the profiles of semi-volatile compounds present in mainstream tobacco smoke particulate matter trapped on glass fibre filters for two types of cigarettes differing only in filter design. In the first cigarette, the filter cavity contained approximately 60 mg of a weakly basic macroporous polystyrene resin cross-linked with divinyl benzene and with surface amine functionality (CR20), whereas in the second cigarette, it was empty. Relative quantitative analysis, chemical identification, and chemical grouping allowed the use of both parametric and non-parametric analyses to identify differences in the chemical composition of the smokes from these cigarettes. The analysis demonstrated that in addition to the selective partial removal of volatile carbonyls and HCN demonstrated previously, CR20 selectively, but incompletely removed 316 compounds from the particulate phase of cigarette smoke, mainly aryl and aromatic hydrocarbons as well as other more volatile species. In contrast, the relative proportion of amines, hydroxylated aromatic compounds and less volatile species was increased in the smoke from the cigarette containing CR20 in the filter. Our findings show that high resolution GC techniques combined with mass spectrometry and chemometric approaches are powerful tools for deconvoluting the complexity of combustion aerosols, as well as helping to identify changes in chemical composition resulting from modifications to cigarette designs. [Beitr. Tabakforsch. Int. 28 (2019) 231–249]

Study of the mainstream cigarette smoke aerosols by Fourier transform ion cyclotron resonance mass spectrometry coupled to laser/desorption and electrospray ionization - Additional insights on the heteroaromatic components.

The chemical composition of the particulate phase of cigarette smoke inhaled by the active smoker is still poorly known in spite of its importance from a health point of view. A non-targeted approach is applied to cigarette smoke particles collected on a quartz filter to obtain an as complete as possible description of this complex mixture. A home-made smoking machine including devices for volatile organic compounds (VOCs) and particle sampling was used. The validation of the cigarette smoking and cigarette smoke collection procedures was conducted by the quantification of some compounds by gas chromatography/mass spectrometry (GC/MS). The particles were investigated by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) directly after their collection on quartz filters by laser/desorption ionization (LDI) or after extraction with CH2 Cl2 by electrospray ionization (ESI). The determination of the benzene, toluene, ethylbenzene and xylenes (from 2 to 35 μg/cigarette) and nicotine (0.68 ± 0.05 mg/cigarette) validated the used sampling method. The complementarity of the LDI and ESI sources for the cigarette smoke analysis was established. The ESI analyses evidenced polar compounds and components with a pyridine group and LDI ensured the detection of poly-condensed heteroaromatic species. Finally, this methodology was employed to characterize particles from cigarettes with or without flavoring additives. Some insights into the composition of cigarette smoke inhaled by active smokers have been obtained. The ~1750 observed features revealed the huge complexity of cigarette smoke particles and the diversity of the possible associated health issues. Both heteroaromatic and highly oxygenated compounds produced by combustion and pyrolysis have been highlighted.

Outcomes of developmental exposure to total particulate matter from cigarette smoke in zebrafish (Danio rerio)

The effects of prenatal exposure to cigarette smoke remain a subject of major interest, especially as it relates to neural development and adverse behavioral outcomes. Several studies have investigated the developmental toxicity of cigarette smoke components in a zebrafish model, showing that developmental exposure to total particulate matter (TPM; particulate phase of cigarette smoke) leads to adverse physiological aberrations and locomotor hyperactivity. Thus, the current study examines whether developmental TPM exposure of zebrafish embryos/larvae (F0) leads to physiological and behavioral alterations, and whether adverse effects are observed in adult fish and the next generation (F1; i.e. F0 offspring). We also examine whether behavioral effects are associated with changes in neural development, stress response, neurotransmitters, and bioenergetics. We demonstrate that TPM exposure during F0 development increased the incidence of deformities in F0 larvae, but F1 larvae did not exhibit any deformities. TPM exposure also resulted in swimming hyperactivity in F0 larvae and several behavioral changes were noted in F0 fish when they grew into adulthood. These behavioral changes were generally not associated with changes in markers of neural development in larvae, stress response in F0 adults, and concentration of neurotransmitters (acetylcholine, dopamine, and serotonin) in F0 adult brain. There were also no changes in F0 or F1 embryonic oxygen consumption rate (OCR; marker of bioenergetics and mitochondrial health); however, the OCR in the brain of F0 males was reduced with TPM. We conclude that developmental exposure to TPM affects larval physiology and induces hyperactive swimming behavior, but these effects do not persist in F1 larvae. Moreover, developmental TPM exposure leads to long-lasting sex-specific behavioral outcomes in the F0 adult fish.

Heterocyclic aromatic amines and their contribution to the bacterial mutagenicity of the particulate phase of cigarette smoke.

Heterocyclic aromatic amines (HAAs) rank among the strongest known mutagens. Approximately 30 HAAs have been found in cooked foods (broiled, fried, and grilled) and several HAAs have been characterized as animal carcinogens. Nine HAAs have also been reported to be constituents of cigarette smoke (CS) raising concerns that HAAs might contribute significantly to the known carcinogenicity of CS. As HAAs are found predominantly in the total particulate matter (TPM) of CS, an improved method for the quantification of HAAs in TPM is reported allowing detection and quantification of 8 HAAs in a single run. The mutagenic potency of these HAAs and that of TPM from the reference cigarette 2R4F was determined in the Salmonella Reverse Mutation Assay (Ames assay) with tester strain TA98 and a metabolic activation system. The 8 HAAs, when applied together in the Ames assay, showed a clear sub-additive response. Likewise, the combination of HAAs and TPM, if at all, gave rise to a slight sub-additive response. In both cases, however, the sub-additive response in the Ames assay was observed at HAA doses that are far above the amounts found in CS. The contribution of the individual HAAs to the total mutagenic activity of TPM was calculated and experimentally confirmed to be approximately 1% of the total mutagenic activity. Thus, HAAs do not contribute significantly to the bacterial in vitro mutagenicity of CS TPM.

Cigarette Smoke-induced Morphological Transformation of Bhas 42 Cells In Vitro

In vitro cell transformation assays detect transformed cells that have acquired the distinct characteristics of malignant cells and thus model one stage of in vivo carcinogenesis. These assays have been proposed as surrogate models for predicting the non-genotoxic carcinogenic potential of chemicals. The Bhas 42 cell transformation assay, a short-term assay that uses v-Ha-ras-transfected Balb/c 3T3 cells, can detect the tumour promoter-like activities of chemicals, but has not previously been used with cigarette smoke. The particulate phase of cigarette smoke (total particulate matter [TPM]) is known to induce tumours in vivo in the mouse skin painting assay. Therefore, we investigated the ability of this Bhas cell assay to form morphologically transformed foci in vitro when repeatedly challenged with TPM from a standard research cigarette. TPM induced a dose-dependent increase in Type III foci, and a significant increase (up to 20-fold) in focus formation at moderately toxic concentrations between 5 and 60µg TPM/ml, with a peak at 20µg/ml. Three batches of TPM were tested in three independent experiments. Precision (repeatability and reproducibility) was calculated by using 0, 5, 10, and 20µg TPM/ml. Repeatability and reproducibility, expressed as the relative standard deviation obtained from the normalised slopes of the dose-response curves, were 17.2% and 19.6%, respectively; the slopes were 0.7402 ± 0.1247, 0.9347 ± 0.1316, and 0.8772 ± 0.1767 (increase factor∗ml/mg TPM; mean ± SD) ; and the goodness of fit (r2) of the mean slopes, each derived from n = 6 repeats, was 0.9449, 0.8198, and 0.8344, respectively. This in vitro assay with Bhas 42 cells, which are regarded as already initiated in the two-stage paradigm of carcinogenesis (initiation and promotion), is able to detect cell transformation induced by cigarette smoke in a dose-dependent manner with a high sensitivity and good precision. Because this assay is fast and yields reliable results, it may be useful in product assessment, as well as for further investigation of the non-genotoxic carcinogenic activity of tobacco smoke-related test substances.

Evaluation of Superoxide Anion Radicals Generated from an Aqueous Extract of Particulate Phase Cigarette Smoke by Electron Spin Resonance Using 5,5-Dimethyl-1-pyrroline-N-oxide

The reactive oxygen species generated by an aqueous extract of the particulate phase of cigarette smoke were evaluated by an electron spin resonance (ESR) analysis, using spin-trapping agents, and by comparing with model reaction systems. The ESR signals of DMPO-OH were detected from the extract by using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). These signals were eliminated by adding superoxide dismutase, but hardly by catalase. These responses of the ESR signals to the scavengers were similar to those of a hypoxanthine-xanthine oxidase system. The results indicate that the signals of DMPO-OH from the extract were derived from a reaction product of DMPO with superoxide anion radicals and clarify the mechanism by which the extract generated superoxide anion radicals.

Analysis of Hydrogen Peroxide in an Aqueous Extract of Cigarette Smoke and Effect of pH on the Yield

An analysis of hydrogen peroxide in an aqueous extract of cigarette smoke, which contains many redox-active compounds, requires a method with high selectivity. An aqueous extract of the particulate phase of cigarette smoke was analyzed by HPLC with an electrochemical detector (ECD). Samples were prepared by collecting the particulate phase of the cigarette smoke on a glass fiber filter and extracting it with a phosphate buffer. The obtained solution was purified by using a Waters Oasis MCX cation-exchange cartridge, and then analyzed by an HPLC-ECD system with a Shodex KS-801 mixed-mode resin column. Pre-injecting hydrogen peroxide at a high concentration into the HPLC instrument stabilized the analytical results. The recovery of hydrogen peroxide by using an extract of the particulate phase of the cigarette smoke was more than 80%. An increase in the amount of hydrogen peroxide was observed during extraction with the phosphate buffer at higher pH values. In contrast, extraction with phosphoric acid did not increase the amount of hydrogen peroxide during extraction.

Particle Size Distribution of Nicotine in Mainstream Smoke from 2R4F, Marlboro Medium, and Quest1 Cigarettes under Different Puffing Regimens

Nicotine’s dose and rate of delivery to the brain play an important role in its addiction and cardiovascular effects. Nicotine is mainly present in the particulate phase of cigarette smoke, and since particle size distribution controls the deposition behavior of particles in the respiratory tract, changes in the particle size distribution can produce variations in its regional and total dose to the lung. These variations can change its absorption rate and delivery to the brain. The particle size distribution of mainstream smoke (MS) varies with changes in puffing regimen and cigarette design and composition. This study examined nicotine in different particle size fractions of MS generated from 2R4F, Marlboro Medium, and Quest1 cigarettes using 3 puffing regimens: (1) FTC-like puff, 35 ml over 2 s; (2) short puff, 50 ml over 2 s; and (3) long puff, 100 ml over 10 s. MS was generated in a chamber at 37°C and>95% relative humidity (RH), and size-segregated particles were collected using RJR cascade impactors. Particle size distribution was determined by spectrophotometry. Nicotine was anayzed using gas chromatography and mass spectrometry. Results showed that nicotine speciates in larger particles (1.1–1.9 μm diameter) under the long puffing regimen and in smaller particles (0.4–1.1 μm diameter) under the short puffing regimen, while mass median aerodynamic diameter of mainstream smoke particles was found to be approximately constant (0.9–1.0 μm) for the three puffing regimens. Overall, changes in puffing regimen have a significant effect on particle size distribution of nicotine and its deposited dose.

Determination of Benzene and Toluene In Exhaled Cigarette Smoke

Abstract This study describes the results regarding the evaluation of retention efficiency by humans of benzene and toluene from cigarette smoke. The evaluated cigarette was a common commercial cigarette with 10.6 mg ‘tar’ [U.S. Federal Trade Commission (FTC) ‘tar’ is defined as the weight of total particulate matter minus nicotine and water]. The test was performed on ten subjects. The exhaled smoke was collected using a vacuum assisted technique that avoids strain in exhaling the smoke. The study showed that benzene was retained at levels of 89% to 98%, and toluene was retained at similar levels, between 87% and 99%. The lower limits of retention for both benzene and toluene are unexpectedly low compared to the retention of bi-and tricyclic aromatic hydrocarbons, which have retentions from cigarette smoke above 95%. This is probably caused by the fact that monocyclic aromatic hydrocarbons are present practically only in the vapor phase of cigarette smoke and at considerably higher levels than bi- and tri-cyclic aromatic hydrocarbons which are present almost completely in the particulate phase of cigarette smoke.

Involvement of Semiquinone Radicals in the in Vitro Cytotoxicity of Cigarette Mainstream Smoke

Free radicals in cigarette smoke have attracted a great deal of attention because they are hypothesized to be responsible in part for several of the pathologies related to smoking. Hydroquinone, catechol, and their methyl-substituted derivatives are abundant in the particulate phase of cigarette smoke, and they are known precursors of semiquinone radicals. In this study, the in vitro cytotoxicity of these dihydroxybenzenes was determined using the neutral red uptake (NRU) assay, and their radical-forming capacity was determined by electron paramagnetic resonance (EPR). All of the dihydroxybenzenes studied were found to generate appreciable amounts of semiquinone radicals when dissolved in the cell culture medium employed in the NRU assay. Hydroquinone exhibited by far the highest capacity to form semiquinone radicals at physiological pH, even though it is not the most cytotoxic dihydroxybenzene. Methyl-substituted dihydroxybenzenes were found to be more cytotoxic than either hydroquinone or catechol. The formation of semiquinone radicals via auto-oxidation of the dihydroxybenzenes was found to be dependent on the reduction potential of the corresponding quinone/semiquinone radical redox couple. The capacity to generate semiquinone radicals was found to be insufficient to explain the variance in the cytotoxicity among the dihydroxybenzenes in our study; consequently, other mechanisms of toxicity must also be involved. The observed interactions between 2,6-dimethylhydroquinone and hydroquinone in the cytotoxicity assay and EPR analysis suggest that care needs to be taken when the bioactivity of cigarette smoke constituents is evaluated, i.e., the effect of the cigarette smoke complex matrix on the activity of the single constituent studied must be taken into consideration.

Toll-like receptor 2 expression is decreased on alveolar macrophages in cigarette smokers and COPD patients

BackroundCigarette smoke exposure including biologically active lipopolysaccharide (LPS) in the particulate phase of cigarette smoke induces activation of alveolar macrophages (AM) and alveolar epithelial cells leading to production of inflammatory mediators. This represents a crucial mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Respiratory pathogens are a major cause of exacerbations leading to recurrent cycles of injury and repair. The interaction between pathogen-associated molecular patterns and the host is mediated by pattern recognition receptors (PRR's). In the present study we characterized the expression of Toll-like receptor (TLR)- 2, TLR4 and CD14 on human AM compared to autologous monocytes obtained from patients with COPD, healthy smokers and non-smokers.MethodsThe study population consisted of 14 COPD patients without evidence for acute exacerbation, 10 healthy smokers and 17 healthy non-smokers stratified according to age. The expression of TLR2, TLR4 and CD14 surface molecules on human AM compared to autologous monocytes was assessed ex vivo using FACS analysis. In situ hybridization was performed on bronchoalveolar lavage (BAL) cells by application of the new developed HOPE-fixative.ResultsThe expression of TLR2, TLR4 and CD14 on AM from COPD patients, smokers and non-smokers was reduced as compared to autologous monocytes. Comparing AM we detected a reduced expression of TLR2 in COPD patients and smokers. In addition TLR2 mRNA and protein expression was increased after LPS stimulation on non-smokers AM in contrast to smokers and COPD patients.ConclusionOur data suggest a smoke related change in the phenotype of AM's and the cellular response to microbial stimulation which may be associated with impairment of host defenses in the lower respiratory tract.

Near-real-time determination of hydrogen peroxide generated from cigarette smoke

The ability to monitor hydrogen peroxide (H2O2) in aqueous smoke extracts will advance our understanding of the relationship between cigarette smoke-induced oxidative stress, inflammation, and disease and help elucidate the pathways by which the various smoke constituents exert their pathogenic effects. We have demonstrated, for the first time, the measurement of H2O2 production from cigarette smoke without prior separation of the sample. Cigarettes were tested on a commercial smoking machine, such that the whole smoke or gas vapor phase was bubbled through phosphate buffered saline solution at pH 7.4. Aliquots of these solutions were analyzed using an Amplex Red/horseradish peroxidase fluorimetric assay that required only a 2 minute incubation time, facilitating the rapid, facile collection of data. Catalase was used to demonstrate the selectivity and specificity of the assay for H2O2 in the complex smoke matrix. We measured approximately 7-8 microM H2O2 from two reference cigarettes (i.e., 1R4F and 2R4F). We also observed 9x more H2O2 from whole smoke bubbled samples compared to the gas vapor phase, indicating that the major constituent(s) responsible for H2O2 formation reside in the particulate phase of cigarette smoke. Aqueous solutions of hydroquinone and catechol, both of which are particulate phase constituents of cigarette smoke, generated no H2O2 even though they are free radical precursors involved in the production of reactive oxygen species in the smoke matrix.

Analysis of polycyclic aromatic hydrocarbons in the particulate phase of cigarette smoke using a gas chromatographic-high-resolution mass spectrometric technique.

A new procedure is developed for the extraction of polycyclic aromatic hydrocarbons (PAHs) from the particulate phase of cigarette smoke. The procedure applies solid-phase extraction using a Bond Elut CH cartridge as a sample preparation step. The efficiency of the cleanup procedure is verified using a gas chromatographic (GC)-high-resolution mass spectrometric (MS) technique, proving that no interference occurs in the PAHs' determination. The efficient cleanup allows GC detection using either high- or low-resolution MS detection. Enhanced sensitivity is obtained using GC-MS and selected ion monitoring. This new technique has several advantages over other reported techniques. The method is simple and robust and has good repeatability and accuracy. The estimated detection limit is 0.1 ng/cigarette for benzo[a]pyrene. In addition to that, the recovery from the smoke pad in which the smoke is collected is approximately 97% for all PAHs. Results for the PAH analyses for 1R5F, 1R4F, and 1R3 Kentucky reference cigarettes are reported in this study. These results provide useful evidence for clarifying the controversy about previously reported data.

A rapid method for the determination of mercury in mainstream cigarette smoke by two-stage amalgamation cold vapor atomic absorption spectrometryPresented at 55th Tobacco Science Research Conference, Greensboro, NC, USA, September 2001.

A rapid method for the determination of mercury in mainstream cigarette smoke was developed utilizing reusable gold–platinum gauze to collect vapor phase mercury. Either an electrostatic precipitator or a Cambridge filter is used to remove the particulate phase of cigarette smoke, followed by a second Cambridge filter to further absorb volatile organic components. The vapor phase mercury is selectively trapped onto the gold–platinum gauze and is then determined by two-stage amalgamation cold vapor atomic absorption spectrometry (CVAAS) without matrix interference. One of the major advantages of this method is the avoidance of lengthy sample preparation procedures, which are required when using liquid impinger traps. Only one cigarette is required for smoke generation because of the preconcentration of mercury before quantitation and the enhanced sensitivity of amalgamation CVAAS. The limit of detection is 0.04 ng. The analytical precision of elemental mercury is 6% at 0.1 ng and 0.3% at 10 ng in terms of relative standard deviation. For the 1R4F reference cigarette smoked under the FTC smoking regime and an alternative smoking regime, the vapor phase mercury in the mainstream smoke is 5.0 ± 0.4 ng per cigt (n = 20) and 7.4 ± 0.4 ng per cigt (n = 10), respectively. The recovery of fortified sample matrix is 97 ± 8% (n = 5) for FTC smoking regime and 96 ± 4% (n = 5) for the alternative smoking regime. The analytical results of the 1R4F reference cigarette were in good agreement with those obtained by the conventional liquid impinger method.

The volatile fraction of cigarette smoke induces alterations in the human gingival fibroblast cytoskeleton.

Several in vitro investigations have indicated that the particulate phase of cigarette smoke, such as nicotine, affects many cell types, including gingival fibroblasts. However, few studies have been performed on the effects of the volatile fraction on the cellular structures that are involved in cell functions, such as adhesion and proliferation. Since the survival and reproduction of gingival fibroblasts are fundamental in maintaining the integrity of the oral connective tissue, as well as in wound healing, the effects on the cytoskeleton of acrolein and acetaldehyde, which are the volatile fractions of cigarette smoke, were examined in vitro for human gingival fibroblasts (HGFs). HGF strains that were taken from healthy subjects with non-inflamed-gingiva were utilized in this investigation. The cells were incubated in the presence of different concentrations of acrolein and acetaldehyde. Cell adhesion and viability were evaluated after incubation for 3 h and 5 days, respectively. The influence on cytoskeletal structures (tubulin, actin and vimentin intermediate filaments) was investigated with the indirect immunofluorescence technique. The results show that both substances produced similar effects, which resulted in a dose-dependent inhibition of HGF adhesion and viability. Disturbance of the HGF cytoskeleton consisted of disruption of the microtubules, actin filaments and vimentin microfilaments, which was accompanied by alterations to cell shape. Our experimental findings suggest that the volatile fractions of cigarette smoke, such as acrolein and acetaldehyde, have a cytotoxic effect on HGFs, with the result that they lose their capacity for adhesion and proliferation. The consequences of this could be impairment of the maintenance, integrity and remodelling of the oral connective tissue. According to our morphological evidence, these findings show that cigarette smoke can lead to the development and progression of periodontal disease, and indicate the need for appropriate therapy.

Human gingival fibroblast cytoskeleton is a target for volatile smoke components.

Several in vitro investigations have indicated that the particulate phase of cigarette smoke as nicotine affects many cell types including gingival fibroblasts, but few studies have examined the effect of volatile fraction on cellular structures involved in cell functions such as adhesion and proliferation. Since gingival fibroblast survival and reproduction are fundamental to maintaining the oral connective tissue as well as to wound healing, the effects of acrolein and acetaldehyde, volatile fractions of cigarette smoke, on cytoskeleton were examined in human gingival fibroblasts (HGFs) in vitro. Human gingival fibroblast (HGF) strains from healthy subjects with non-inflamed gingiva were utilized. The cells were incubated in different concentrations of acrolein and acetaldehyde. Cell adhesion was evaluated after 3 hours. The influence of both substances on cytoskeletal structures, tubulin and vimentin intermediate filaments (VIF), was investigated using indirect immunofluorescence technique. The results show that both substances produced similar effects, resulting in a dose-dependent inhibition of HGF adhesion. Disturbance of HGF cytoskeleton consisted of a disruption of microtubules and vimentin microfilaments with alterations in cell shape. Our experimental findings suggest that volatile fractions of cigarette smoke such as acrolein and acetaldehyde, because their ability to bind and interact with the cytoskeleton, prevent HGF adhesion. Consequently the maintenance of the oral connective tissue and integrity and remodeling could be impaired. According to our morphological evidence, these findings confirm other clinical and epidemiological investigations reporting that volatile components of cigarette smoke could lead to the initiation and progression of periodontal disease.

Determination of Dibenzacridines in the Particulate Phase of Cigarette Smoke

Abstract This study attempted to resolve a controversy related to the presence of dibenz[a,j]acridine and dibenz[a,h]acridine in the particulate phase of cigarette smoke. Smoking was performed using FTC conditions (35 mL puff volume, 2 sec. puff, 1 min. interval) on a Borgwaldt RM 20/CS smoking machine. The particulate phase of forty cigarettes was collected on 92 mm Cambridge filter pads. Pads were combined to analyze the particulate phase of the mainstream smoke from between 120 and 320 cigarettes. In an initial scheme of analysis, the pads were extracted with an acidic aqueous solution. This aqueous solution was then washed with CH2Cl2 and the organic phase discarded. The aqueous solution was then changed to basic and extracted with CH2Cl2, which was concentrated and analyzed via GC/MS. The dibenzacridine could not be detected utilizing this scheme, even when the pads had been spiked with a few thousand nanograms of dibenzacridine. After using several other organic solvents (cyclohexane, CHCl3, and benzene) to eliminate the possibility that the extraction efficiency of CH2Cl2 was poor, it was determined that dibenzacridine was being discarded with the first CH2Cl2 wash. A successful separation scheme was developed by extracting the smoked pad with an aqueous acidic solution, followed by extraction of the aqueous phase with CH2Cl2without pH change. The CH2Cl2 extract was concentrated under nitrogen and 1 µL injected for GC/MS analysis. Quantification was achieved by spiking the pads with dibenz[a,j]acridine-d13 as an internal standard at a level equal to 1.7-2.5 ng/cig. The limit of detection for this technique was approximately 0.5 ng/cig. The chromatographic separation was performed with a 30 m BPX-5 column (0.25 mm i.d., 0.25 µm film thickness). Mass spectral data were acquired in selected ion monitoring (SIM) mode with m/z = 279 for the two dibenzacridine isomers and m/z = 292 for the deuterated internal standard. Three commercial cigarettes were analyzed: two commercial Full Flavor cigarettes, one commercial Light cigarette, and a 1R4F reference cigarette. Neither dibenzacridine isomer was detected in any of the samples analyzed.

Effects of nicotine on the immune response. I. Chronic exposure to nicotine impairs antigen receptor-mediated signal transduction in lymphocytes.

Previous work has demonstrated that chronic exposure of rats to cigarette smoke causes inhibition of the antibody-forming cell (AFC) response and that the particulate phase of cigarette smoke, containing most of the nicotine in cigarette smoke, is essential for immunosuppression. Using intradermally implanted miniosmotic pumps, LEW rats were exposed to nicotine or its principal metabolite, cotinine, at the rate of about 14 micrograms/hr for 3-4 weeks. Serum cotinine levels in nicotine-treated (NT) animals of 219 +/- 40 ng/ml (on Day 10) were comparable to average human smokers. No significant differences between control (CON) and NT animals were observed in the distribution of lymphocyte subsets. However, nicotine, but not cotinine, treatment for 3 to 4 weeks inhibited both the T-dependent and T-independent AFC responses and proliferation to anti-CD3. Con A response was observed in 4-week but not in 3-week NT animals. Cell cycle analysis revealed that upon stimulation with Con A or anti-CD3, in spite of comparable surface expression of IL-2 receptors and class II MHC molecules, significantly fewer NT T cells entered the S and G2/M phases than CON T cells, indicating an arrest in the G0/G1 phase. Furthermore, B and T cells from NT animals were unable to elevate the intracellular calcium levels normally in response to ligation of antigen receptors, although Ca2+ responses of salivary gland cells to acetylcholine were normal. Thus, nicotine may significantly contribute to the immunosuppressive effects of chronic smoking by inducing a state of anergy in lymphocytes and may be related to their impaired response to antigen-induced signaling.

Rapid method for the determination of benzo[a]pyrene in the particulate phase of cigarette smoke by high-performance liquid chromatography with fluorimetric detection.

A method is described for the rapid determination of benzo[a]pyrene in the particulate phase of cigarette smoke. The particulate matter from five cigarettes is fractionated on Florisil and a concentrated fraction injected on to a high-performance liquid chromatography column of Partisil. Benzo[a]pyrene is detected in the eluate fluorimetrically. Quantification is achieved by means of a standard-additions method.

Studies on the effect of irritant vapors on ciliary mucus transport: I. Phenol and cigarette smoke

A simple reproducible method for the study of the effects of tobacco smoke and other materials upon frog cilia was developed. This method consists of the measurement of mucus flow rates in preparations of frog esophageal tissue, in the presence of 0.00625% sodium adenosine triphosphate (ATP). The presence of ATP during the measurements allows the use of 100% of the frog esophageal preparations, prevents the complete arrest of mucus flow after repeated exposure of frog tissue preparations to cigarette smoke, assures remarkably constant and reproducible mucus flow rates for periods of time extending up to 24 hours, and renders possible conditions of exposure of frog cilia to tobacco smoke simulating human smoking. With the aid of this technique it was shown that smoke from a filter cigarette has considerably less cilia-inhibitory effect than smoke from a nonfilter cigarette. This effect is of high statistical significance and is attributed, at least in part, to selective removal of phenols from the smoke by the cigarette filter. Vapors of pure phenol had little or no effect on ciliary activity. In the presence of smoke from a filter cigarette, however, phenol became a markedly cilia-inhibiting agent, probably in conjunction with the particulate phase of cigarette smoke. On the other hand, phenol vapors did not potentiate the effect of smoke from a nonfilter cigarette, and it was concluded that smoke from nonfilter cigarettes is already saturated with phenol.