Abstract

Although the involvement of environmental tobacco smoke (ETS) in human lung cancer is no longer a matter of dispute, the magnitude of its impact still is. This is mainly due to the inefficiency of methodology to assess exposure to ETS especially in public places. Setting a real life exposure condition (3 h stay in local pubs) and using a matched-control study design, we quantified smoke-related DNA adducts in induced sputum and peripheral blood lymphocytes (PBL) of healthy non-smokers ( n = 15) before and after a single pub visit by means of the 32 P-post-labeling assay. For verification, we also measured a spectrum of polycyclic aromatic hydrocarbons (PAH) in the ambient air of the pubs by personal air monitors, and determined the plasma concentrations of nicotine and cotinine by gas chromatography/mass spectrometry. The ambient air concentrations of all PAH were several orders of magnitude higher than those already reported for other indoor environments. The plasma concentrations of both nicotine and cotinine increased significantly after the pub visit ( P = 0.001 and P = 0.0007, respectively). Accordingly, the overall DNA adduct profile in induced sputum, but not in PBL, changed quantitatively and qualitatively after the pub visit. Of most significance was the formation of a distinct DNA adduct in induced sputum of three individuals consequent to ETS exposure. This adduct co-migrated with the standard (±)- anti -benzo[ a ]pyrene diol epoxide–DNA adduct, which is known to form at lung cancer mutational hotspots. We conclude that real life exposure to ETS can give rise to pro-mutagenic lesions in the lower airway, and this can be best investigated in a relevant surrogate matrix such as induced sputum.

Introduction

Accumulating evidence links exposure to environmental tobacco smoke (ETS) to lung cancer ( 1,2 ). Yet, the magnitude of risk for ETS-related lung cancer is not well established (reviewed in ref. 3). This uncertainty arises mainly from the methodological inefficiency in assessing exposure to ETS. Understandingly, the profile of exposure to ETS is highly complicated because ETS emanates from multiple origins, in varying compositions and concentrations. Also, the time frame of ETS exposure is quite variable ( 4 ). Ideally, exposure assessment for ETS would involve specific and sensitive measurements of ETS in the environment as well as in the internal milieu of the body ( 4 ). The former can be achieved by monitoring the ambient air for various ETS constituents, whereas the latter can be done by dosimetry of these constituents in the body fluids or by quantifying them after their interaction with subcellular targets, e.g. DNA, RNA and proteins ( 5,6 ).

Polycyclic aromatic hydrocarbons (PAH) are a major class of carcinogens present in ETS ( 7 ) and have been implicated in the etiology of several human cancers including cancer of the lower respiratory tract ( 8 ). The carcinogenicity of PAH is mostly ascribed to their specific chemical structures, which enable them to bind covalently to DNA and form DNA adducts ( 9 ). Formation of DNA adducts is an event of potential significance in carcinogenesis because it may give rise to mutagenic events leading to chromosomal aberrations, DNA strand breaks, oncogene activation and tumor suppressor gene inactivation ( 10–12 ). Thus, DNA adduct formation not only represents a prior exposure to carcinogens but it may also indicate a tangible risk for cancer ( 10 ). Theoretically, dosimetry of DNA adducts should be performed within the organs where the tumor arises (target organs). Practically, however, most target organs for PAH-associated cancers are only invasively accessible. Also, the commonly used non-target organs show inconsistent surrogacy for the target ones ( 13 ). This latter might relate to the incomparability of exposure patterns in surrogate versus target organs. In addition, different cell compositions with varying biotransformational and DNA repair capacity in target versus surrogate organs may also be responsible for such inconsistency ( 14,15 ). Recently, we have shown the applicability of induced sputum, a non-invasively obtainable surrogate matrix from the lower airway, for dosimetry of smoke-related DNA adducts ( 16,17 ). Here, we utilized DNA adduct dosimetry in induced sputum and in a conventional surrogate matrix, peripheral blood lymphocytes (PBL), for assessing exposure to ETS. Thus, in a group of healthy non-smokers who volunteered to be exposed to ETS in local pubs, we quantified aromatic/hydrophobic–DNA adducts in induced sputum and PBL using the 32 P-post-labeling assay, before and after a single pub visit. To complement our DNA adduct dosimetry data, we also measured the concentrations of a spectrum of PAH in the ambient air of the pubs by personal air monitors. For verification, we quantified the internal doses of two well-documented ETS markers, nicotine and its main metabolite, cotinine ( 18,19 ), in plasma at both pre- and post-exposure times by gas chromatography/mass spectrometry (GC/MS).

Material and methods

Study population

Volunteers were Dutch students of the Faculty of Health Sciences, Maastricht University, Maastricht, The Netherlands. Upon enrollment, each individual filled out a questionnaire on his/her age, gender, active and passive smoking status, alcohol consumption, dietary intake, medical history of disease and familial history of cancer. Subsequently, 15 healthy non-smokers who had no considerable exposure to ETS either at home or outside, were selected and signed informed consents. As part of our study protocol, they all abstained from visiting a pub, nightclub or any smoky site at least 2 weeks prior to the experiment. The study population consisted of 11 females and four males with an average age of 22 ± 1.5 years (mean ± SD). For logistic reasons, they were subdivided in three groups of four individuals and one group of three individuals. The study was approved by the Medical Ethical Commission of Maastricht University.

Study protocol

Before pre-sampling, all participants were interviewed in detail and briefed on the study protocol. After collection of peripheral blood and sputum, subjects wore a personal air monitor and then went to the local pubs in their assigned groups. To rule out the inter-group bias, all groups visited specific pubs at certain nights of the week when there were student gatherings. The subjects spent ∼3 h in a relatively highly smoky area of the pub whilst drinking only non-alcoholic beverages; they were supervised by a staff member throughout. In case that there was a drop in the crowd density of the pub, they were instructed to quickly move into a busier pub in the neighborhood. Immediately after the pub visit, all subjects were transported to the study center and post-sampled for peripheral blood and sputum. In all cases, the lag time between exposure ending and post-sampling was within 1–2 h. All samples were coded for further laboratory analyses.

Personal air monitoring

Personal breathing zone air samples were collected by a SKC plastic cyclone (SKC, Eighty Four, PA), connected to a pre-charged pocket pump at a flow rate of 2.2 l/min. The unit collects respirable particulates with an aerodynamic diameter of 4.0 μm onto a teflon PTFE filter (pore size, 2.0 μm) (Omega Specialty Instrument, Chelmsford, MA) at a 50% collection efficiency ( 20 ). The particulates were extracted from the filters by sonication with acetonitrile and subsequently, analyzed for a spectrum of PAH by high-performance liquid chromatography with fluorescence detection ( 20 ).

Peripheral blood

Twenty milliliters of venous blood was drawn into a heparinized Venoject ® II tube (Terumo Europe N.V., Leuven, Belgium). Lymphocyte and plasma fractions were separated using the LeucoSep ® tubes (Greiner GmbH, Frickenhausen, Germany) according to the instructions of the manufacturer. Isolated PBL and plasma were stored at –70°C until further analysis.

Induced sputum

Sputum induction and processing were done as described earlier ( 17 ). Briefly, after pre-treatment with inhalatory salbutamol (200 μg), subjects inhaled ultrasonically nebulized 4.5% saline delivered from an Ultra-Neb™ 2000 (De Vilbiss, Somerset, PA) for a period of 3×7 min with 5 min intervals. During the intervals, subjects rinsed their mouth, gargled the throat and then coughed up the produced expectorate into a 50 ml Greiner tube (Greiner GmbH) placed on ice. Additionally, they were instructed to cough up the available expectorate at any moment irrespective of the time of induction. Induction was terminated at the end of 21 min of inhalation, or as soon as sufficient amount of sputum (5 ml) was obtained. Sputolysis processing of the samples was done according to our published protocol ( 16 ). The processed samples were divided into two portions, one for DNA isolation and the other for cytological examination.

DNA isolation

Isolation of DNA was performed according to a standard protocol ( 16 ) using phenol–chloroform extraction and ethanol precipitation techniques.

Gas chromatography/mass spectrometry

Concentrations of nicotine and cotinine in plasma were measured by GC/MS with heavy isotope dilution as described by Jacob et al . ( 21 ). Samples of 100 μl were added to 10 ng of D 4 -nicotine, and 100 ng of D 4 -cotinine in 0.01 M HCl. After addition of 50 μl 2 M NaOH containing 0.2 M ammonia and extraction with toluene/1-butanol (70:30, 300 μl) the organic layer was transferred to tubes containing 50 ml 0.5 M sulfonic acid. After freezing in a dry ice–acetone bath the organic layer was discarded. Aqueous potassium carbonate (50 ml of 50 w/v containing 0.2 M ammonia) was added to the aqueous phase and the samples were extracted with 30 μl toluene–butanol [90:10]. One microliter of the organic phase was injected in the splitless mode on the HP 5 MS fused silica capillary column of 12.0 m length and 0.25 mm inner diameter. A Hewlett-Packard 6890 Series GC (Palo Alto, CA) with 5973 mass selective detector was used. The carrier gas was helium with a column flow of 1 ml/s. The temperature gradient of the column oven was 70°C for 1 min, 12°C/min increase for 8.5 min followed by a 30°C/min increase for 3.7 min until 270°C and kept for 3 min. Selective ion monitoring was 84 and 88 m/z for nicotine and D 4 -nicotine and 176 and 180 m/z for cotinine and D 4 -cotinine, respectively.

32 P-post-labeling assay

The 32 P-post-labeling assay was carried out as described earlier ( 17 ). Briefly, DNA (5 μg) was digested into deoxyribonucleoside 3′-monophosphates by incubation with micrococcal endonuclease (0.5 U) and spleen phosphodiesterase (3.3 μg) for 3 h at 37°C. Half of the digest was treated with NP1 (7.3 μg) for 40 min at 37°C. The reaction was terminated by adding 0.5 M Tris and labeling was performed using AT-1γ- 32 P (50 μCi) in the presence of T4-polynucleotide kinase (5.8 U) for 30 min at 37°C. Subsequently, thin-layer chromatography was done on polyethyleneimine (PEI)–cellulose sheets (Macherey Nagel, Düren, Germany) with the following solvent systems: D 1 , 1 M NaH 2 PO 4 , pH 6.5; D 2 , 8.5 M urea, 5.3 M lithium formate, pH 3.5; D 3 , 1.2 M lithium chloride, 0.5 M Tris, 8.5 urea, pH 8.0; D 4 , 1.7 M NaH 2 PO 4 , pH 6.0. Detailed information on the efficiency of NP1 digestion, ATP excess, and nucleotide quantification are available in ref. ( 16 ). Quantification was done using a PhosphorImager (Molecular Dynamics™, Sunnyvale, CA) with a lower detection limit of 1 adduct/10 9 nucleotides. For standardization, two samples of [ 3 H](±)- anti -benzo[ a ]pyrene diol epoxide (BPDE) with known modification levels of 1 adduct/10 7 and 10 8 unmodified nucleotides were run in parallel in all experiments.

Statistical analysis

Results were expressed as mean ± standard error. All variables at pre-exposure time were compared with their respectives at post-exposure time by the Wilcoxon signed rank test. Relationships between different variables were explored by the Spearman rank correlation analysis. Statistical significance was considered at P ≤ 0.05.

Results

Ambient air concentrations of PAH and personal dosimetry data

The results of personal air monitoring in each group as well as overall groups of this study population are summarized in (Table I ). The amount of individual or total PAH extracted from the ETS-exposed filters was considered as the respective respirable dose. In all cases, the respirable doses were linearly related to the total volume of air flown into the filters. Thus, we adjusted the respirable dosimetry data for air volume. Consequently, the coefficients of variation for respirable doses of individual and total PAH were as follows: benzo[ a ]anthracene, 27%; chrysene, 23%; benzo[ b ]fluoranthene, 23%; benzo[ k ]fluoranthene, 19%; benzo[ a ]pyrene (B[ a ]P), 22%; total PAH, 21%. For quality control, air samples were taken from two different indoor environments (a living room and an office) for the same duration of time and with identical devices. In neither case, was there any detectable individual or total PAH in the extracted filters.

Plasma nicotine and cotinine

The distribution of the levels of plasma nicotine and cotinine in all individuals at pre- and post-exposure times ( T0 and T1 , respectively) is shown in (Figure 1A and B ). Plasma nicotine levels ranged from 0.72 to 1.03 ng/ml at T0 and from 1.09 to 1.51 at T1 ( P = 0.001). Although plasma cotinine was only measurable in one individual at T0 , all individuals had quantifiable levels of plasma cotinine at T1 varying in the range of 1.72–3.92 ng/ml ( P = 0.0007). Overall, there was no significant difference in the internal doses of nicotine or cotinine among different groups of the study population (coefficients of variation for nicotine and cotinine levels were 11.3 and 24.6%, respectively). The internal doses of cotinine were positively but non-significantly related to the respirable doses of individual as well as total PAH.

Success rate for sputum induction

Sputum induction was well tolerated by all subjects and no troublesome symptoms occurred throughout the procedure. Thirteen of the 15 individuals at pre-exposure time and 14 of them at post-exposure time could produce sufficient amount of sputum for further analysis (Table II ).

Cytological examinations and DNA yield of induced sputum

Cellular characteristics of induced sputum samples at pre- and post-exposure times ( T0 and T1 , respectively) are presented in Table II . Overall, there was no significant difference in induced sputum cytology at T0 as compared with T1 ; total cell count (7.7 ± 1.9 versus 6.8 ± 2.0×10 6 ; P = 0.4); cell viability (73.1 ± 4.3 versus 79.5 ± 5.4%; P = 0.3); non-squamous cells (82.8 ± 4.0 versus 89.0 ± 2.7%; P = 0.09). The overall non-squamous cell composition was as follows, bronchoalveolar macrophages, 80 ± 7%; neutrophils, 11 ± 2%; bronchoepithelial cells, 8.5 ± 2.1%; lymphocytes, 0.3 ± 0.1%; metachromatic cells, 0.2 ± 0.1%. Also, DNA yield of induced sputum samples at T0 did not differ from that at T1 (29.4 ± 5.9 versus 35.1 ± 7.8 μg; P = 0.5).

DNA adducts in induced sputum and PBL

The representative chromatograms of the 32 P-post-labeled DNA adducts from an individual at pre- and post-exposure times ( T0 and T1 , respectively) are shown in Figure 2A and B . Overall, the DNA adduct profile consisted of three distinct spots (spots 1, 2 and 3) and a faint diagonal radioactive zone (DRZ) (Table II ). The most clear-cut signal, spot 1, was present only at T1 and co-migrated with standard BPDE–DNA adduct (Figure 2B and C ). The other two spots and the DRZ were observed both at T0 and T1 . Qualitatively, spot 1 was observed in the adduct maps of three individuals at T1 . Spot 2 was seen in the adduct maps of four and two individuals at T0 and T1 , respectively. Spot 3 was the most prevalent one and appeared in the adduct maps of 10 and 11 individuals at T0 and T1 , respectively. The respective number of individuals at T0 and T1 who showed a DRZ in their adduct maps, were three and four. Quantitatively, among those who displayed the spots 2 or 3 or DRZ at both time points, paired comparison showed no significant change in the levels of either of these two spots or the DRZ at T1 as compared with T0 . Nonetheless, there was a marginal enhancement in the overall spots and DRZ (considered as the total level of adducts) at T1 as compared with T0 (1.57 ± 0.69 versus 1.40 ± 0.44/10 8 nucleotides, P = 0.7). The total level of adducts varied in the ranges of 0.28–4.1/10 8 nucleotides at T0 and 0.30–4.5/10 8 nucleotides at T1 . Within the individuals, there was no association between the spots 1, 2, 3 and DRZ either at T0 or T1 . For verification, induced sputum samples from three known smokers were analyzed in parallel as well. All the three revealed an intense DRZ in their adduct maps and had a total adduct level of 2.92 ± 0.9/10 8 nucleotides (Figure 2D ).

Overall, there was no detectable level of adducts in PBL of most individuals either at T0 or at T1 ; only three samples had fairly quantifiable DNA adducts (just above the detection limit of our assay) with no correlation with induced sputum DNA adducts. Furthermore, there was no relationship between DNA adduct dosimetry data in induced sputum or PBL and the respirable doses of individual or total PAH, or the internal doses of nicotine/cotinine.

Discussion

The experimental setting of this study was chosen to mimic the individual's real life exposure to ETS. Our personal air monitoring data indicate that spending an average period of time in a smoky pub results in a considerably high exposure to carcinogenic ETS. We observed relatively high concentrations of a spectrum of PAH in the ambient air of all pubs where our experiments were conducted. These concentrations were notably higher than those reported previously for other indoor environments. For instance, in our study the mean concentration of B[ a ]P, a widely known representative of carcinogenic PAH ( 7 ), was 6.3 ng/m 3 . The mean concentrations of this compound in smoking and non-smoking homes were 1.0 and 0.4 ng/m 3 , respectively ( 22 ). Also, the respective values in commercial buildings were 1.07 ng/m 3 for smoking zones and 0.39 ng/m 3 for non-smoking zones ( 23 ). The reported mean concentration of B[ a ]P in workplaces such as coke plants was within the range of 0.35–0.63 μg/m 3 ( 24 ). In the ambient air of a highly polluted industrial city, Silesia in Poland, the mean concentrations of B[ a ]P were 60–90 ng/m 3 in winter and 5–20 μg/m 3 in summer ( 25 ).

In confirmation, our internal dosimetry data of nicotine showed the absorption and metabolism of this well-documented ETS marker ( 18,19 ) after the pub visit. In vivo , nicotine is rapidly metabolized (half life, 2 h) to more stable metabolites, such as nicotine-1- N -oxide, nornicotine and cotinine ( 18,19,26 ). Of these, cotinine (half life, 21–48 h in non-smokers; 27) is by far the most utilized marker in the studies of ETS ( 28 ). It has already been shown that the steady state of cotinine predicts the daily intake of nicotine as every 100 ng/ml plasma cotinine represents a daily intake of 8 mg of nicotine ( 29 ). In the present study, the mean increase in the concentration of plasma cotinine after the putative exposure to ETS was 2.4 ng/ml. This corresponds with an intake of 0.197 mg of nicotine, which is an approximate equivalent of the nicotine intake resulting from smoking 7.7% of a non-filter cigarette ( 30 ). Interestingly, our personal air monitoring data revealed a mean respirable dose of 2.1 ng B[ a ]P, which is present in the amount of 20–40 ng per non-filter cigarette ( 30 ), therefore corresponding with smoking 5.2–10.5% of the cigarette. From the standpoint of methodology, our pre-exposure data of nicotine could also verify the compliance of the study population with the protocol as they showed that all participants had maintained a non-ETS exposure profile prior to the experiment.

Likewise, our dosimetry of aromatic/hydrophobic–DNA adducts in induced sputum underlined distinguishable enhancement in DNA adduct profiles after the pub visit. Apparently, the 3 h putative exposure in the pubs and the subsequent 1–2 h lag time for post-sampling was sufficient to give rise to the formation of ETS-derived DNA adducts in the induced sputum cells. Of most significance was a specific exposure-related DNA adduct (spot 1), which appeared in the adduct maps of three individuals at post-exposure time. This adduct co-migrated with the standard BPDE–DNA adduct that has been shown to form preferentially at p53 mutational hotspots ( 11 ). The p53 is mutated in 60% of human lung cancers and its mutational spectra differ between smokers and non-smokers in that smokers have a significant increase in G to T transversions (reviewed in ref. 31). Recently, the G to T transversion hotspots in LacI , cII and SupF transgenes have been shown to correlate with the preferential binding sites of BPDE at methylated CpG dinucletotides in these genes ( 32 ). Also, in vitro treatment of human bronchial epithelial cells with BPDE induced the frequency of G to T transversions at codons 157, 248 and 249 of the p53 , all designated mutational hotspots in lung cancer ( 33 ). Given these established links between BPDE–DNA adduct and lung cancer, the herein appearance of this adduct consequent to the pub visit supports a causality between ETS and lung cancer. It would be of interest to identify this DNA adduct in non-smoking lung cancer patients with a known history of exposure to ETS. An alternative approach would be to repeat our experiment in healthy non-smokers with familial history of lung cancer. In both cases, analysis of DNA adducts should preferably be performed in the lung tissue or induced sputum cells. Previously, Finette et al . ( 34 ) had demonstrated a significant difference in hypoxanthine-guanine phosphoribosyltransferase ( HPRT ) mutational spectra, but not mutant frequency, in cord blood lymphocytes from newborns exposed via placenta to ETS, compared with their non-exposed counterparts ( 34 ). The single most striking alteration was an increase in `illegitimate' genomic HPRT deletion mediated by V(D)J recombinase in the exposed newborns ( 34 ). As the `illegitimate' mutational events mediated by V(D)J recombinase have been associated with pediatric lymphoid malignancy ( 35,36 ), this report was the first demonstration of the in utero involvement of ETS in carcinogenesis. However, the V(D)J recombinase-mediated mutations may also occur spontaneously, or as a result of exposure to other environmental factors, such as pesticides ( 37–39 ). Compared with that report, our study is privileged to have a more direct approach owing to its matched-control experimental design. Also, the herein quantified endpoints and in particular, the BPDE–DNA adduct are all exclusively relevant for lung cancer. To our knowledge, this is the first time that an experimental study can establish a plausible link between ETS and lung cancer.

Moreover, there were two other notable adducts (spots 2 and 3) and a faint DRZ in the adduct maps of induced sputum samples. However, they were all non-specific as they were present at both pre- and post-exposure times and changed only marginally after the pub visit. In addition, within the individuals the two spots and DRZ did not associate with each other either before or after the pub visit. Altogether, it appears that the quantified adducts and DRZ originate from the putative exposure in the pub as well as the ubiquitous exposure in the environment. Although the enhancement of most adducts after the pub visit is to be expected, the disappearance of adduct 2 and the decrease in the level of adduct 3 may well be due to the repair of DNA and/or cell turn over.

Conversely, DNA adduct analysis in PBL produced no conclusive results as there were almost no quantifiable DNA adducts in this cell type either before or after experimental exposure to ETS. This is not, however, surprising because previous studies by others ( 40,41 ) also failed to demonstrate any increase in DNA adduct levels in PBL or total white blood cells after exposure to ETS. Also, applying a much more intense and controlled exposure condition (8 h in a confined chamber), Holz et al . ( 42 ) found similar results in peripheral blood monocytes. Taken together, it seems that the total or fractionated blood cells are not a choice of preference for the surrogacy of an organ like the lung. Understandingly, the circulating blood cells are exposed to multiple sources of carcinogens, which enter the body via different routes, e.g. inhalation, ingestion and dermal absorption. In addition, they have varying biotransformational and DNA repair capacity and various life spans ( 43–45 ). Thus, they may reflect an integrated DNA adduct burden for the entire body but not for an individual organ.

Lastly, our DNA adduct dosimetry data were indirectly confirmed by personal air monitoring and internal dosimetry data, which showed the presence of carcinogenic PAH in the ambient air of the pubs, and the absorption of nicotine in all exposed individuals, respectively. Albeit, there was no linear relationship between external/internal dosimetry data and those of DNA adducts in induced sputum or PBL. We should, however, acknowledge that exposure to carcinogen is only a preliminary step in carcinogenesis, whereas DNA adduct formation is an intermediate one. Unlike carcinogen exposure, formation of DNA adducts can be affected by various parameters that are different within and between the individuals. Hence, it is conceivable that the external dosimetry and DNA adduct data may not correspond to each other especially in small study populations. With the same token, the lack of relationship between adduct dosimetry data and the internal dosimetry data of nicotine, which had already been reported by others ( 45,47 ) is not unexpected. Besides, biotransformation of nicotine involves a different pathway (mainly catalyzed by CYP2A6 and CYP↑2D6; 48) from that of the herein DNA adduct inducing agents.

We summarize that real life exposure to ETS has the potential to trigger the formation of pro-mutagenic lesions in the lower respiratory tract, and this is best demonstrated in a valid surrogate matrix such as induced sputum. From the standpoint of public health, our findings warrant further investigations to evaluate the health consequences of exposure to ETS in different indoor environments including the occupational settings.

Table I.

Summary data of personal air monitoring

  Group I ( n = 3)   Group II ( n = 4)   Group III ( n = 4)   Group IV ( n = 4)   Overall group (n = 15)  
  Concentrations (ng/m 3 )  Dose (ng)  Concentrations (ng/m 3 )  Dose (ng)  Concentrations (ng/m 3 )  Dose (ng)  Concentrations (ng/m 3 )  Dose (ng)  Concentrations (ng/m 3 )   Dose a (ng)  
Results are expressed as mean ± SEM. 
a Coefficients of variation for respirable doses of benzo[ a ]anthracene, 27%; chrysene, 23%; benzo[ b ]fluoranthene, 23%; benzo[ k ]fluoranthene, 19%; benzo[ a ]pyrene, 22%; total PAH, 21%.  
Benzo[ a ]antracene  13.2 ± 1.6 4.3 ± 0.5 10.7 ± 1.4  3.4 ± 0.5 12.9 ± 1.5  4.4 ± 0.5 12.6 ± 2.5  4.5 ± 0.9 12.3 ± 0.9 4.2 ± 0.3 
Chrysene 18.5 ± 0.3 6.1 ± 0.1 14.7 ± 1.5  4.7 ± 0.5 19.8 ± 1.5  6.7 ± 0.5 19.4 ± 3.1  7.0 ± 1.1 18.1 ± 1.1 6.1 ± 0.4 
Benzo[ b ]fluoranthene  4.9 ± 0.8 1.6 ± 0.3 3.8 ± 0.1  1.2 ± 0.03 5.0 ± 0.4  1.7 ± 0.2 4.5 ± 0.7  1.6 ± 0.2 4.5 ± 0.3 1.5 ± 0.1 
Benzo[ k ]fluoranthene  1.2 ± 0.1 0.4 ± 0.1 0.9 ± 0.1  0.3 ± 0.02 1.0 ± 0.1  0.3 ± 0.04 1.1 ± 0.1  0.4 ± 0.03 1.0 ± 0.1 0.4 ± 0.1 
Benzo[ a ]pyrene  6.8 ± 0.3 2.2 ± 0.1 6.0 ± 0.5  1.9 ± 0.2 6.5 ± 1.0  2.2 ± 0.3 6.2 ± 1.0  2.2 ± 0.4 6.3 ± 0.4 2.1 ± 0.1 
Total PAH 44.6 ± 3.0 14.7 ± 1.0 36.1 ± 2.8 11.6 ± 0.9 45.2 ± 4.1 15.3 ± 1.3 43.8 ± 6.4 15.7 ± 2.3 42.3 ± 2.2 14.3 ± 0.8 
Total air flown into the filter (l)  173 ± 1.0 168 ± 0.3  178 ± 0.9 188 ± 0.3  177 ± 7.9     
  Group I ( n = 3)   Group II ( n = 4)   Group III ( n = 4)   Group IV ( n = 4)   Overall group (n = 15)  
  Concentrations (ng/m 3 )  Dose (ng)  Concentrations (ng/m 3 )  Dose (ng)  Concentrations (ng/m 3 )  Dose (ng)  Concentrations (ng/m 3 )  Dose (ng)  Concentrations (ng/m 3 )   Dose a (ng)  
Results are expressed as mean ± SEM. 
a Coefficients of variation for respirable doses of benzo[ a ]anthracene, 27%; chrysene, 23%; benzo[ b ]fluoranthene, 23%; benzo[ k ]fluoranthene, 19%; benzo[ a ]pyrene, 22%; total PAH, 21%.  
Benzo[ a ]antracene  13.2 ± 1.6 4.3 ± 0.5 10.7 ± 1.4  3.4 ± 0.5 12.9 ± 1.5  4.4 ± 0.5 12.6 ± 2.5  4.5 ± 0.9 12.3 ± 0.9 4.2 ± 0.3 
Chrysene 18.5 ± 0.3 6.1 ± 0.1 14.7 ± 1.5  4.7 ± 0.5 19.8 ± 1.5  6.7 ± 0.5 19.4 ± 3.1  7.0 ± 1.1 18.1 ± 1.1 6.1 ± 0.4 
Benzo[ b ]fluoranthene  4.9 ± 0.8 1.6 ± 0.3 3.8 ± 0.1  1.2 ± 0.03 5.0 ± 0.4  1.7 ± 0.2 4.5 ± 0.7  1.6 ± 0.2 4.5 ± 0.3 1.5 ± 0.1 
Benzo[ k ]fluoranthene  1.2 ± 0.1 0.4 ± 0.1 0.9 ± 0.1  0.3 ± 0.02 1.0 ± 0.1  0.3 ± 0.04 1.1 ± 0.1  0.4 ± 0.03 1.0 ± 0.1 0.4 ± 0.1 
Benzo[ a ]pyrene  6.8 ± 0.3 2.2 ± 0.1 6.0 ± 0.5  1.9 ± 0.2 6.5 ± 1.0  2.2 ± 0.3 6.2 ± 1.0  2.2 ± 0.4 6.3 ± 0.4 2.1 ± 0.1 
Total PAH 44.6 ± 3.0 14.7 ± 1.0 36.1 ± 2.8 11.6 ± 0.9 45.2 ± 4.1 15.3 ± 1.3 43.8 ± 6.4 15.7 ± 2.3 42.3 ± 2.2 14.3 ± 0.8 
Total air flown into the filter (l)  173 ± 1.0 168 ± 0.3  178 ± 0.9 188 ± 0.3  177 ± 7.9     
Table II.

Sputum induction, cytology, DNA yield and DNA adduct dosimetry at pre- and post-pub visit

 Pre-sampling Post-sampling 
nt, Nucleotides. 
a Results are expressed as mean ± SEM. For comparative analyses, only paired values are considered.  
b Number of samples positive (+) for the respective adduct spots are indicated.  
c This adduct co-migrated with standard BPDE–DNA adduct.  
d Not detected.  
Sputum induction (success/failure) 13/2 14/1 
Total cell count (×10 6 ) a  7.7 ± 1.9  6.8 ± 2.0 
Viability (%) a 73.1 ± 4.3 79.5 ± 5.4 
Non-squamous cells (%) a 82.8 ± 4.0 89.0 ± 2.7 
DNA yield (μg) a 29.4 ± 5.9 35.1 ± 7.8 
Qualitative DNA adducts b   
    Spot 1 c  ND 4 
    Spot 2 
    Spot 3 10 11 
    DRZ 
Quantitative DNA adducts a   
    Spot 1 (/10 8 nt)   ND d 0.29 ± 0.04 
    Spot 2 (/10 8 nt)  0.40 ± 0.14 0.59 ± 0.27 
    Spot 3 (/10 8 nt)  0.69 ± 0.17 0.52 ± 0.13 
    DRZ (/10 8 nt)  2.29 ± 0.7 2.37 ± 1.51 
    Total (/10 8 nt)  1.40 ± 0.44 1.57 ± 0.69 
 Pre-sampling Post-sampling 
nt, Nucleotides. 
a Results are expressed as mean ± SEM. For comparative analyses, only paired values are considered.  
b Number of samples positive (+) for the respective adduct spots are indicated.  
c This adduct co-migrated with standard BPDE–DNA adduct.  
d Not detected.  
Sputum induction (success/failure) 13/2 14/1 
Total cell count (×10 6 ) a  7.7 ± 1.9  6.8 ± 2.0 
Viability (%) a 73.1 ± 4.3 79.5 ± 5.4 
Non-squamous cells (%) a 82.8 ± 4.0 89.0 ± 2.7 
DNA yield (μg) a 29.4 ± 5.9 35.1 ± 7.8 
Qualitative DNA adducts b   
    Spot 1 c  ND 4 
    Spot 2 
    Spot 3 10 11 
    DRZ 
Quantitative DNA adducts a   
    Spot 1 (/10 8 nt)   ND d 0.29 ± 0.04 
    Spot 2 (/10 8 nt)  0.40 ± 0.14 0.59 ± 0.27 
    Spot 3 (/10 8 nt)  0.69 ± 0.17 0.52 ± 0.13 
    DRZ (/10 8 nt)  2.29 ± 0.7 2.37 ± 1.51 
    Total (/10 8 nt)  1.40 ± 0.44 1.57 ± 0.69 
Fig. 1.

Distribution of plasma concentrations of nicotine ( A ) and cotinine ( B ) in the overall study population at pre- and post-exposure times.

Fig. 1.

Distribution of plasma concentrations of nicotine ( A ) and cotinine ( B ) in the overall study population at pre- and post-exposure times.

Fig. 2.

Representative chromatograms of the 32 P-post-labeled DNA adducts in induced sputum of a non-smoker at pre- and post-exposure times ( A and B , respectively), the BPDE–DNA adduct standard (1 adduct/10 8 nucleotides) ( C ) and a known smoker ( D ). Spots 1, 2 and 3 are encircled and the DRZ is bordered with two lines. The radioactivity in the upper left-hand corner was also observed at the same location in non-modified calf thymus DNA and therefore, considered as background.

Fig. 2.

Representative chromatograms of the 32 P-post-labeled DNA adducts in induced sputum of a non-smoker at pre- and post-exposure times ( A and B , respectively), the BPDE–DNA adduct standard (1 adduct/10 8 nucleotides) ( C ) and a known smoker ( D ). Spots 1, 2 and 3 are encircled and the DRZ is bordered with two lines. The radioactivity in the upper left-hand corner was also observed at the same location in non-modified calf thymus DNA and therefore, considered as background.

5
To whom correspondence should be addressed Email: ania@coh.org

The authors wish to thank Prof. Dr Gerd P. Pfeifer (Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA) for critically reviewing the manuscript and giving valuable comments on it. Special thanks to Drs Peyton Jacob III and Neal Benowitz (University of California, San Francisco, CA) for their kind gifts of deuterium labeled nicotine M +4 and cotinine M +4 for GC/MS analysis.

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