Comparison between smoking-related DNA adduct analysis in induced sputum and peripheral blood lymphocytes
A.Besarati Nia,
L.M. Maas,
E.M.C. Brouwer,
J.C.S. Kleinjans and
F.J. Van Schooten1
Department of Health Risk Analysis and Toxicology, Maastricht University, PO Box 616, 6200 MD, Maastricht, The Netherlands
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Abstract
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We investigated the applicability of induced sputum (IS), a non-invasive derivative from the lower respiratory tract, for smoking-related DNA adduct analysis and its comparability with peripheral blood lymphocytes (PBL). Lipophilic DNA adducts were quantified by the 32P-post-labeling assay in IS and PBL of smokers (n = 9) with stable smoking status at three time points (one week intervals) and non-smokers (n = 9) at one time point. The success rate for sputum induction was 100% at all time points. There was no significant difference in total cell count, cell viability, squamous cell count and DNA yield between smokers and non-smokers. Within the smokers, there was no significant difference in IS cytology at the three time points: overall (mean of three measurements) total cell count, 9.0 ± 2.4x106; cell viability, 77 ± 4%; squamous cell count, 28 ± 5%; non-squamous cell count, 72 ± 4% (bronchoalveolar macrophages, 75 ± 6%; neutrophils, 17 ± 3%; bronchoepithelial cells, 7 ± 2%; lymphocytes, 0.7 ± 0.2%; metachromatic cells, 0.3 ± 0.2%). IS DNA yield did not differ significantly at the three time points [overall (mean of three extractions) DNA yield, 66 ± 20 µg]. A typical smoking-associated diagonal radioactive zone was observed in the adduct maps of IS and PBL of all and five smokers, respectively, and of none of the non-smokers. Lipophilic DNA adduct levels in both IS and PBL of smokers were higher than those of non-smokers (3.7 ± 0.9 versus 0.7 ± 0.2/108 nt, P = 0.0005, and 2.1 ± 0.3 versus 0.6 ± 0.1/108 nt, P = 0.0001, respectively). In smokers the level of adducts in IS was non-significantly higher than that in PBL (3.7 ± 0.9 versus 2.1 ± 0.3/108 nt, P = 0.1), whilst in non-smokers the difference was not appreciable (0.7 ± 0.2 versus 0.6 ± 0.1/108 nt). Within the smokers there was no significant change in the level of adducts at the three time points either in IS or in PBL (coefficients of variation 34 and 29%, respectively). Adduct levels in IS at each time point were higher than those in PBL, leading to a significantly higher overall (mean of three quantifications) level of adducts in IS than PBL (3.3 ± 0.2 versus 2.1 ± 0.1/108 nt, P = 0.02). The overall levels of adducts in both IS and PBL were dose-dependently related to smoking indices. We conclude that IS is a preferable matrix as compared with PBL for molecular dosimetry of (current) exposure to inhalatory carcinogens as its analysis reveals both the existence and the magnitude of exposure more explicitly.
Abbreviations: CI, confidence interval; CV, coefficient of variation; DRZ, diagonal radioactive zone; IS, induced sputum; PBL, peripheral blood lymphocytes; PBS, phosphate-buffered saline; PEI, polyethyleneimine.
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Introduction
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To date, DNA adducts are known as biologically effective dose markers of exposure to carcinogens as well as potential markers of cancer susceptibility (1,2). So far, DNA adduct analysis has been performed in various biological matrices, of which peripheral blood lymphocytes (PBL) are the most utilized (36). Owing to their accessibility in a relatively non-invasive fashion and their capability of reflecting DNA adduct formation consequent to exposure to carcinogens (7), PBL have been used as a surrogate matrix in numerous investigations (36). However, as circulating blood cells being exposed to multiple DNA adduct-inducing agents entering the body via diverse routes, e.g. ingestion, inhalation and absorption, PBL mainly represent the integrated DNA adduct burden for the entire body and not for an individual organ (8). This questions their validity as a surrogate for those organs which are exposed to specific carcinogens via particular routes. For instance, the observed discrepancy between DNA adduct analysis in PBL and lung tissue (913) throws doubt upon the representativeness of PBL for portraying the events occurring in the lung, a primary target site for most inhalatory carcinogens.
As part of our ongoing project to identify innovative biological materials which can serve for biomonitoring of putatively exposed humans, we have embarked on research on induced sputum (IS), a non-invasively obtainable matrix from the lower respiratory tract. Thus far, we have reported the applicability of IS for molecular dosimetry of exposure to inhalatory carcinogens (14) by demonstrating significantly higher levels of smoking-related DNA adducts in IS of smokers as compared with non-smokers. Here, we validated our previous findings and made a comparison between DNA adduct analysis in IS and PBL. Further, we assessed the consistency of DNA adduct analysis at the intra-individual level in IS as well as in PBL. To achieve these objectives, we quantified lipophilic DNA adducts in IS and PBL of a group of smokers with stable smoking status at three consecutive time points by means of the 32P-post-labeling assay applying nuclease P1 as the enrichment method. For comparison purposes, we also measured the respective levels of adducts in IS and PBL of a control group of non-smokers at time point 1.
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Materials and methods
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Study population
Healthy smoking and non-smoking volunteers who participated in our previous study (14) were re-recruited (one participant who had quit smoking since then was also included, being considered as an ex-smoker; she was, however, excluded from the statistical analyses). Characteristics of the study population are listed in Table I
. The study population consisted of a group of smokers (n = 9) with an average age of 38 ± 5 years and a group of lifelong non-smokers (n = 9) aged 34 ± 3 years. Prior to sampling, all participants were interviewed to re-check their available database and briefed for the study protocol; they were advised to strictly maintain their lifestyles, particularly smoking behavior and dietary intake, throughout the study. Smoking status was evaluated using current smoking indices (number of cigarettes smoked per day and amount of tar consumed per day) and smoking history index (pack years). Tar consumption per day was calculated as the number of cigarettes smoked per day multiplied by the tar content (mg) of the cigarette. Pack years was estimated as the number of cigarettes smoked per day divided by 20 and multiplied by smoking years. As the smoking status of most of the participants had changed since the previous study, we could not use the then obtained data (as time point 1 data) for comparison purposes. The study outline was three consecutive samplings of blood and sputum at intervals of 1 week in the smoking group and a single sampling of blood and sputum in the non-smoking group. The study protocol was approved by the Medical Ethical Commission of Maastricht University.
Peripheral blood
Twenty milliliters of venous blood was drawn into heparinized Venoject® II tubes (Terumo Europe NV, Leuven, Belgium). Lymphocyte isolation was done according to the method of Bøyum (15) by gradient centrifugation of the samples over LymphoprepTM (Nycomed Pharma, Oslo, Norway). Isolated lymphocytes were pelleted and preserved at 80°C until DNA isolation.
Induced sputum
Induction of sputum and processing of the samples were done as described earlier (14). Briefly, after pretreatment with inhalatory salbutamol (200 µg), subjects inhaled ultrasonically nebulised 4.5% saline delivered from an Ultra-NebTM 2000 (De Vilbiss, Somerset, USA) device for a period of up to 21 min. There were three 5 min intervals at the end of each 7 min inhalation period. During the intervals, subjects rinsed their mouths, gargled and then coughed up the produced expectorate into a 50 ml Greiner tube (Greiner Labortechnik, Frickenhausen, Germany) 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 a sufficient amount of sputum (5 ml) was obtained. IS samples were processed by adding 0.1% Sputolysine (Calbiochem-Novabiochem Corp., La Jolla, USA) (equal to 4 vol of sample), followed by incubation in a shaking water bath for 15 min at 37°C. The samples were intermittently vortexed for 15 s and aspirated with a 25 ml pipette. To quench the activity of the Sputolysine, 4 vol of phosphate-buffered saline (PBS), pH 7.4, were added and incubation continued for another 5 min. The resulting homogenates were centrifuged at 725 g for 10 min at 4°C. Supernatants were discarded and the pellets resuspended in 2 ml of PBS from which 100 µl aliquots were used for cytological examination and the remainders were re-pelleted to be preserved at 80°C until DNA isolation. Determinations of cell viability according to the trypan blue exclusion technique and total cell counts were carried out using 10 µl of the cell suspensions in a standardized hemocytometer. From the remaining cell suspensions, aliquots of 30x103 cells (diluted in PBS to a final volume of 300 µl) were cytocentrifuged (Shandon, Cheshire, UK) at 1500 r.p.m. for 5 min onto PolysineTM microslides (E.Merck Nederland BV, Amsterdam, The Netherlands). The slides were stained with May-Grünwald Giemsa and cell differentiation was determined by counting 500 non-squamous cells/slide.
DNA isolation
The DNA contents of both IS and PBL were extracted as described earlier (14). Briefly, cell pellets were thawed and then lysed with 400 µl SET/SDS (100 mM NaCl, 20 mM EDTA, 50 mM Tris, 0.5% SDS, pH 8.0) at 37°C overnight. The resulting suspensions were treated with 50 µl RNase mixture (0.1 mg/ml RNase A and 1000 U/ml RNase T1) for 3 h at 37°C, followed by treatment with 75 µl proteinase K (10 mg/ml) for 2 h at 37°C. DNA was isolated by repetitive extraction with phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1) and then precipitated with 2 vol of 100% cold ethanol and 1/30 vol of 3 M sodium acetate, pH 5.3. Precipitated DNA was rinsed with 70% ethanol and dissolved in 2 mM Tris, pH 7.4. Quality and quantity of the DNA were determined spectrophotometrically (A230/260 ~0.43, A260/280 ~1.8) and ultimately its concentration was adjusted to 2 mg/ml.
32P-post-labeling assay
The 32P-post-labeling assay was performed as described earlier (14). Briefly, 10 µg of DNA was digested to deoxyribonucleoside 3'-monophosphates using calf spleen phosphodiesterase (2 µg/µl) and micrococcal endonuclease (0.25 U/µl). Half of the digest was treated with nuclease P1 (2.5 g/µl) and subsequently labeled with [
-32P]ATP in the presence of T4 polynucleotide kinase. Radiolabeled adducted nucleotide biphosphates were separated by two-dimensional chromatography on polyethyleneimine (PEI)cellulose sheets (Macherey Nagel, Düren, Germany) using the following solvent systems: D1, 1 M NaH2PO4, pH 6.5; D2, 8.5 M urea, 5.3 M lithium formate, pH 3.5; D3, 1.2 M lithium chloride, 0.5 M Tris, 8.5 urea, pH 8.0; D4, 1.7 M NaH2PO4, pH 6.0. To ensure the efficiency of nuclease P1 treatment and ATP excess, an aliquot of the digest was one-dimensionally chromatographed on PEIcellulose sheet (Merck, Darmstadt, Germany) using a solvent system of 0.12 M NaH2PO4, pH 6.8. For quantification purposes, two standards of [3H]7ß,8
-dihydroxy-9
,10
-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene modified nucleotides (1 adduct per 107 and 108 unmodified nucleotides) were run in parallel in all experiments. Quantification was performed using phosphorimaging technology (Molecular DynamicsTM, Sunnyvale, USA). Quantitatively, half of the detection limit for the diagonal radioactive zone (DRZ) (0.25 adducts/108 nucleotides) was considered as the determined level of adducts for samples which showed neither a DRZ nor an adduct spot in their adduct maps. Nucleotide quantification was done by labeling the remaining half of the digested DNA with [
-32P]ATP in the presence of T4 polynucleotide kinase and by subsequent separation of the nucleotides by one-dimensional chromatography on PEIcellulose sheet using the solvent system of 0.12 M NaH2PO4, pH 6.8.
Statistical analysis
Results are expressed as means ± SEM throughout the text. The MannWhitney U-test was used to make a comparison between smokers and non-smokers for all variables in both IS and PBL at time point 1. Friedman's two way analysis of variance was performed to assess the intra-individual variations of all variables over time in both IS and PBL of smokers; coefficients of variation (CV) together with 95% confidence intervals (95% CI) are given as indicators of the variability. The Wilcoxon signed rank test was also used to compare the means of each variable (at three time points) in IS with the respective value in PBL. Kendall's rank correlation was utilized to study the relationships between different variables. The ex-smoker was excluded from all analyses. Statistical significance was considered at P < 0.05.
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Results
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Success rate for sputum induction
Sputum induction was 100% successful, as all subjects tolerated the procedure well, did not experience any troublesome symptoms throughout and produced sufficient amounts of sputum for further analysis.
Cytological examinations and DNA yield of induced sputum
Cellular characteristics and DNA yields of IS are presented in Table II
. At time point 1 there was no significant difference in total cell count, cell viability and squamous cell count between smokers and non-smokers (6.5 ± 1.6 versus 7.4 ± 2.8x106, P = 0.7, 74 ± 7 versus 61 ± 8%, P = 0.2, and 31 ± 8 versus 24 ± 8% P = 0.4, respectively). With respect to non-squamous cell differentials, smokers had a significantly higher percentage of neutrophils and a lower percentage of bronchoalveolar macrophages as compared with non-smokers (19 ± 14 versus 5 ± 2%, P = 0.03, and 71 ± 8 versus 92 ± 2%, P = 0.001, respectively). There was no significant difference in DNA yield between smokers and non-smokers (39 ± 10 versus 46 ± 14 µg, P = 0.8) whilst in both smokers and non-smokers the DNA yields were related to the respective total cell counts (r = 0.5, P = 0.08 and r = 0.7, P = 0.01, respectively).
Within the smokers there was no significant difference in total cell count, cell viability and cell differentials at the three time points (Table II
): coefficients of variation in total cell count, cell viability, squamous cell and non-squamous cell counts were 45% (95% CI 2861), 15% (95% CI 624), 18% (95% CI 1125) and 24% (95% CI 931), respectively, and overall (mean of three measurements) total cell count, cell viability, squamous cell and non-squamous cell counts were 9.0 ± 2.4x106, 77 ± 4%, 28 ± 5% and 72 ± 4%, respectively. The overall non-squamous cell differentials were as follows: bronchoalveolar macrophages, 75 ± 6%; neutrophils, 17 ± 3%; bronchoepithelial cells, 7 ± 2%; lymphocytes, 0.7 ± 0.2%; metachromatic cells, 0.3 ± 0.2%. Further, there was no significant change in DNA yield at the three time points (CV 42%, 95% CI 2065); overall (mean of three extractions) DNA yield was 66 ± 20 µg. In addition, the DNA yield at each time point was related to the respective total cell count (r1 = 0.5, P1 = 0.08; r2 = 0.6, P2 = 0.04; r3 = 0.6, P3 = 0.04), resulting in a significant relationship between the overall DNA yield and the overall total cell count (r = 0.6, P = 0.03).
32P-post-labeling of lipophilic DNA adducts
At time point 1, a typical smoking-associated DRZ was observed in the adduct maps of IS of all smokers (but not the ex-smoker). However, the DRZ was only present in the adduct maps of PBL of five smokers. In non-smokers, no DRZ could be seen in the adduct maps of either IS or PBL. Quantitatively, smokers had significantly higher levels of lipophilic DNA adducts in both IS and PBL as compared with non-smokers (3.7 ± 0.9 versus 0.7 ± 0.2/108 nt, P = 0.0005, and 2.1 ± 0.3 versus 0.6 ± 0.1/108 nt, P = 0.0001 respectively). Adduct levels in smokers ranged from 1.4 to 7.1/108 nt in IS and from 1.3 to 2.9/108 nt in PBL. The levels of adducts in non-smokers varied in the ranges 0.251.6/108 nt in IS and 0.251.2/108 nt in PBL. In smokers the levels of adducts in IS were non-significantly higher than those in PBL (3.7 ± 0.9 versus 2.1 ± 0.3/108 nt, P = 0.1), however, in non-smokers the difference was far less pronounced (0.7 ± 0.2 versus 0.6 ± 0.1/108 nt). Only in smokers was there a significant correlation between the level of adducts in IS and PBL (r = 0.8, P = 0.05).
Within the smokers there was a DRZ in the adduct maps of IS of all individuals (but not the ex-smoker) at all time points. The DRZ was only present in the adduct maps of PBL of five individuals (62.5%) at all time points (irrespective of smoking status) (Figure 1
). Neither in IS nor in PBL did lipophilic DNA adduct levels change significantly at the three time points (CV 34%, 95% CI 2049 and CV 29%, 95% CI 355, respectively) (Figure 2
); the levels of adducts in IS at each time point were higher than that in PBL (IS13, 3.7 ± 0.9, 3.4 ± 0.4 and 3.1 ± 0.5/108 nt; PBL13, 2.1 ± 0.3, 2.3 ± 0.8 and 1.9 ± 0.6/108 nt), leading to a significantly higher overall (mean of three quantifications) level of adducts in IS than PBL (3.3 ± 0.2 versus 2.1 ± 0.1/108 nt, P = 0.02) (Figure 3
). The overall levels of adducts ranged from 2.0 to 5.0/108 nt in IS and from 0.9 to 3.5/108 nt in PBL (Figure 3
). Unlike at time point 1, at the other two time points there was no significant correlation between the level of adducts in IS and the respective level in PBL (r2 = 0.4, P2 = 0.3; r3 = 0.5, P3 = 0.2). Nevertheless, the overall level of adducts in IS was significantly related to that in PBL (r = 0.6, P = 0.05). There were doseresponse relationships between the overall levels of adducts in IS and current smoking indices (cigarettes/day, r = 0.6, P = 0.02; tar/day, r = 0.6, P = 0.05). The overall levels of adducts in PBL were dose-dependently related to both current smoking indices (tar/day, r = 0.7, P = 0.02; cigarettes/day, r = 0.6, P = 0.05) and cumulative smoking index (pack years, r = 0.8, P = 0.05, adjusted for tar/day).

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Fig. 1. Representative chromatograms of 32P-post-labeled DNA adducts in IS and PBL of a smoker at three time points. T=1, time point 1; T=2, time point 2; T=3: time point 3.
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Fig. 2. Intra-individual variation in lipophilic DNA adduct levels in IS and PBL of smokers. T=1, time point 1; T=2, time point 2; T=3, time point 3.
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Fig. 3. Lipophilic DNA adduct levels in IS and PBL of smokers and non-smokers over time. Numbers in parentheses indicate time points. IS (M) and PBL (M) represent the mean (of three time points) of adduct levels in IS and PBL, respectively. The lower and upper edges of the boxes are the 25th and the 75th percentiles, respectively. The black ellipses and the lines within the boxes are the means and the medians, respectively. The lower and upper bars are the 10th and the 90th percentiles, respectively. Individual values below the 10th or above the 90th percentiles are shown as . aStatistically significant as compared with non-smoker PBL (1), P = 0.0005. bStatistically significant as compared with non-smoker IS (1), P = 0.0001. cStatistically significant as compared with smoker PBL (M), P = 0.02.
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Discussion
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The absolute success rate for sputum induction and the consistency in cytological examination of IS shown in this study confirmed our previous observation that sputum induction is a feasible, reproducible and reliable technique for sampling lower respiratory tract secretions (14). Analogously, we observed a balance between neutrophil and bronchoalveolar macrophage percentages in both smokers and non-smokers and a significantly higher percentage of neutrophils and lower percentage of bronchoalveolar macrophages in smokers as compared with non-smokers (14). The latter could be ascribed to the fact that inhaled smoking materials might induce inflammation in the airway wherein neutrophils function as the first line of inflammatory cells (14,16,17). We have also noticed that individuals undergoing sputum induction repeatedly produce real sputum with a low level of salivary contamination and a high cell content more easily over time as they learn how to cope with the induction procedure. This could explain the herein intra-individual variation in DNA yield of IS, which was significantly related to total cell count.
Our lipophilic DNA adduct analysis in IS of smokers and non-smokers was also in good agreement with that of our previous report (14), reaffirming the applicability of IS for molecular dosimetry of exposure to inhalatory carcinogens. Similar to that report, we found a DRZ, an indicator of exposure to a complex mixture of chemical carcinogens, e.g. tobacco smoke (18,19), in the adduct maps of IS of all smokers and none of the non-smokers. Further, we observed a significantly higher level (5.3-fold) of adducts in IS of smokers as compared with non-smokers (14). It is worth mentioning that these comparative findings in IS were more striking than the respective findings in PBL, as in the latter case there was only a DRZ in the adduct maps of five of the smokers and the levels of adducts in smokers were 3.5-fold higher than those in non-smokers. It needs to be noted that since most of the non-smokers had hardly detectable level of adducts in either IS or PBL, we excluded this group of individuals from the repeated measurements.
In repeated measurements of lipophilic DNA adducts in IS and PBL of smokers we observed intra-individual stability in DNA adduct analysis in IS as well as in PBL. This gave rise to the idea that a single quantification of adducts in both matrices is valid for at least a short period of time as long as the exposure variable remains constant. We found a DRZ in the adduct maps of IS of all individuals and of PBL of only 62.5% (five out of eight) of the individuals at all time points. The discrepancy in DRZ manifestation between these two matrices could be due to the lower and indirect exposure of PBL to inhalatory carcinogens. Also, IS and PBL, being composed of different cell types, might vary in their capacity to activate/deactivate pro-carcinogens and to repair modified DNA (2023). The absence of a DRZ in the IS adduct map of the ex-smoker who had quit smoking a year ago was not unexpected, as the majority of the cells in IS are short lived with a lifespan of hours to days (2426), thus formation of DNA adducts in IS can predominantly be explained by acute exposure to carcinogens. Moreover, we quantified elevated levels of adducts in IS as compared with PBL at each time point, leading to a 1.6-fold higher overall (mean of three quantifications) level of adducts in IS than PBL. These comparative findings in smokers together with the above-mentioned findings in smokers versus non-smokers are of importance, as the ultimate goal of analyzing DNA adducts in small scale studies like this one is to verify its applicability for exposure assessment in the general population (27). In principle, the analysis should be performed on a biological material where in both the existence and the magnitude of the exposure are explicitly manifest, particularly in the case that the population under investigation is passively exposed to low levels of environmental and/or occupational DNA adduct-inducing agents. Taken together, our data suggest that IS is preferable to PBL for such analysis since, for a given exposure, formation of DNA adducts in IS is more readily distinguishable than that in PBL, hence, we may consider IS as a suitable matrix to be used for biomonitoring of exposure to inhalatory carcinogens.
As mentioned earlier, there was an ex-smoker in our study population whose IS adduct map did not reveal any DRZ. Going through this individual's quantitative data, we noticed that the overall adduct level in her IS had drastically decreased since smoking cessation (from 5.0 to 1.9/108 nt), returning almost to the non-smokers' range established in this study as well as a previous one (14). However, she had an overall adduct level of 1.6/108 nt in her PBL, which was still within the smokers' range (0.93.5/108 nt). This could imply the persistence of adducts in PBL, which have a lifespan of several years (28), suggesting that the detected adducts in PBL at present might have been formed due to chronic exposure to carcinogens. The determined level of adducts in her IS, however, could be considered as a background level of adducts, attributable to environmental exposure to carcinogenic compounds and/or passive smoking (29).
Lastly, there were doseresponse relationships between the overall level of adducts in IS and current smoking indices, indicated by the number of cigarettes smoked per day and the amount of tar consumed per day. On the other hand, the overall levels of adducts in PBL were not only dose-dependently related to the current smoking indices but also to the cumulative smoking index (pack years). It should be noted that although one might argue the validity of these indices as compared with internal dose markers, e.g. urinary cotinine, for assessing exposure to tobacco smoke (30), their reliability upon administration of a standardized questionnaire has already been verified (3133). After all, these doseresponse relationships reiterate the implication that formation of DNA adducts in IS and PBL reflect acute and chronic exposure, respectively, to carcinogens.
In summary, we conclude that IS is a choice of preference for molecular dosimetry of (current) exposure to inhalatory carcinogens as both the existence and the magnitude of exposure can be unequivocally determined in its analysis. Prospectively, IS could be of value in intervention studies where modulation of DNA damage is to be investigated through measurement of the biomarkers of interest.
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Notes
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1 To whom correspondence and reprint requests should be addressed Email: fvanschooten{at}grat.unimaas.nl 
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Received December 13, 1999;
revised March 21, 2000;
accepted March 27, 2000.