Comparison of multiple DNA adduct types in tumor adjacent human lung tissue: effect of cigarette smoking*

R. Godschalk1, J. Nair1,4, F.J. van Schooten2, A. Risch1, P. Drings3, K. Kayser3, H. Dienemann3 and H. Bartsch1

1 German Cancer Research Center (DKFZ), Division of Toxicology and Cancer Risk Factors, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany,
2 University of Maastricht, Department of Health Risk Analysis and Toxicology, Universiteitssingel 50, 6200 MD Maastricht, The Netherlands and
3 Thoraxklinik Heidelberg-Rohrbach, Amalienstraße 5, 69126 Heidelberg, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cigarette smokers inhale a broad range of carcinogens derived from tobacco and its pyrolysis products, including free radicals, which induce oxidative stress and subsequent lipid peroxidation (LPO). Miscoding carcinogen–DNA adducts are formed by cigarette smoke constituents and are thought to initiate lung carcinogenesis. The presence of various types of DNA damage was therefore analyzed in tumor adjacent uninvolved lung tissues of 13 smoking and 11 non-smoking operated lung cancer patients. O4-ethylthymidine (O4etT), 1,N6-ethenodeoxyadenosine ({varepsilon}dA) and 3,N4-ethenodeoxycytidine ({varepsilon}dC) were determined by immuno-enriched 32P-postlabeling. Polycyclic aromatic hydrocarbon (PAH)–DNA adducts were measured as diagonal radioactive zones after nuclease P1 enriched 32P-postlabeling. Mean O4etT and PAH–DNA adduct levels were higher in lung DNA of smokers than of non-smokers (O4etT/108 thymidine: 3.8 versus 1.6, P < 0.01; PAH–DNA adducts/108 nucleotides: 11.2 versus 2.2, P < 0.01). Pulmonary etheno–DNA adduct levels did not differ between smokers and non-smokers, but large inter-individual variations were observed (80- and 250-fold differences for {varepsilon}dA and {varepsilon}dC, respectively). As all smokers (except one) refrained from smoking at least for 1 week before surgery, our results demonstrate the persistence of O4etT and PAH–DNA adducts in human lung. A positive correlation obtained between O4etT and PAH–DNA adducts (R = 0.65, P < 0.01) suggests that both adducts are formed from cigarette smoke as the main exposure source. We conclude that in addition to the DNA adducts derived from PAH and tobacco-specific nitrosamines, miscoding O4etT lesions are formed by cigarette smoke that contribute to the increased genomic instability and increased lung cancer risk in smokers.

Abbreviations: BPDE, benzo[a]pyrene-diol epoxide; {varepsilon}dA, 1,N6-ethenodeoxyadenosine; {varepsilon}dC, 3,N4-ethenodeoxycytidine; LPO, lipid peroxidation; NDEA, N-nitrosodiethylamine; O4etT, O4-ethylthymidine; PAH, polycyclic aromatic hydrocarbons; PEI, polyethyleneimine


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cigarette smoking is causally associated with the development of pre-neoplastic biochemical changes in human lung (1) and lung cancer (2). It is hypothesized that the mechanism of tobacco smoke-induced lung cancer varies according to the location and histological type of the tumor. The deposition of cigarette smoke particulates containing polycyclic aromatic hydrocarbons (PAH) in the major lung bronchi probably initiates the formation of squamous cell carcinoma at that site. Many researchers have focused on the role of PAH-induced DNA damage in the induction of lung cancer (36). The reactive metabolite of the well studied benzo[a]pyrene, benzo[a]pyrene-diol epoxide (BPDE), was found to bind to a specific hotspot in the p53-gene, which has been found to be frequently mutated in human lung cancer (3) and aromatic–DNA adduct levels were found to correlate with lung cancer risk in a recent prospective molecular epidemiological study (4).

On the basis of decreasing concentrations of tar that contains benzo[a]pyrene and other PAH, and increasing levels of alkylating compounds such as tobacco-specific nitrosamines (TSNA) in cigarette smoke, it is plausible that alkylating carcinogens are partially responsible for the rise in the incidence of lung adenocarcinoma, which has now surpassed squamous cell carcinoma as the leading type of lung cancer (7). Tobacco smoke-derived alkylating compounds covalently bind to DNA in the lung and their mutagenic potency depends mainly on the position of binding within the four DNA bases: O-substitution by alkylating agents results in relevant DNA adducts in terms of mutagenesis and carcinogenesis, including O6-alkylguanine and O4-alkylthymine (8). Cigarette smoke contains ethylating compounds, such as N-nitrosodiethylamine (NDEA) and hitherto uncharacterized ethylating agents, since increased levels of N-ethylated DNA bases have been reported in urine of smokers as compared with non-smokers (911). Animal studies indicated that after exposure to ethylating compounds, O4-ethylthymidine (O4etT) is initially formed in very low levels, but is poorly repaired and thus accumulates to biologically relevant levels (12). Our newly developed method for the detection of O4etT made it now possible to study the formation of O4etT in human lung of smoking and non-smoking subjects (13).

The role of continuous exposure to tobacco smoke-derived free radicals in the onset or progression of lung cancer still needs further attention. Free radicals may enhance lipid peroxidation (LPO) in the lung by which reactive unsaturated aldehydes are generated (14). Further oxidation of the aldehyde 4-hydroxy-2-nonenal leads to the formation of 2,3-epoxy-4-hydroxynonanal, a bifunctional alkylating agent which can react with DNA to yield etheno-DNA adducts, including 1,N6-ethenodeoxyadenosine ({varepsilon}dA) and 3,N4-ethenodeoxycytidine ({varepsilon}dC). Etheno–DNA adducts are highly miscoding lesions in mammalian cells and are thought to initiate the carcinogenic process through specific point mutations, as shown for the known carcinogen vinyl chloride (15). However, no information is available yet on the presence and levels of these etheno–DNA adducts in human lung.

In the present study, a direct comparison has been made between the persistence of DNA adducts by different classes of tobacco smoke-derived carcinogens in lung tissue of smoking and non-smoking lung tumor patients. We investigated by 32P-postlabeling, the formation of aromatic–DNA adducts, O4etT, and adducts formed by LPO products, {varepsilon}dA and {varepsilon}dC in tumor adjacent, uninvolved lung tissue of smoking and non-smoking operated lung tumor patients.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Characteristics of study-subjects
Adjacent non-tumorous lung tissues from 13 smoking and 11 non-smoking lung tumor patients (mean age 61 ± 9, 15 male/nine female) were collected at surgery for lung tumor resection. Histological types of lung cancer were determined by the hospital pathologists. Tissue samples were frozen in liquid nitrogen immediately after surgery, and stored at –80°C. Smoking information was obtained from the patient by questionnaire. The study was approved by the ethical committee of the University of Heidelberg (no. 182/96). The overall characteristics of the study subjects are given in Table IGo.


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Table I. Overall characteristics of the study-subjects
 
DNA isolation procedure from human lung samples
Approximately 80 mg of tissue was homogenized in lysis-buffer (DNA-isolation kit supplied by Qiagen, Hilden, Germany) using a polytron homogenizer (15 000 r.p.m. for ~30 s). Proteinase K and RNases were added, followed by a 2 h incubation at 50°C. Subsequently, DNA was isolated using Qiagen columns according to the manufacturer’s protocol with the following modification of the supplied elution buffer; pH was set to 7.4 and the NaCl concentration was increased to 1.4 M. DNA was precipitated by the addition of 0.7 volume isopropanol, collected by centrifugation, washed twice with 70% ethanol and dried in vacuo. Before analysis, DNA was redissolved in water and quantified by spectrophotometry at 260 nm.

Adduct analyses by 32P-postlabeling
O4-ethylthymidine. O4etT was analyzed according to Godschalk et al. (13). Briefly, DNA (25 µg) was digested into nucleotide monophosphates by micrococcal nuclease and spleen phosphodiesterase followed by immunoprecipitation of O4etT with saturated ammonium sulfate using the specific antibody ER-01. Normal nucleotides were quantified by HPLC-UV analysis of the supernatants after adduct precipitation. The antibody–adduct interaction product was collected by centrifugation, redissolved in 400 µl water and transferred into a microcon centrifugation filter (MY-50, molecular weight cut-off: 50 000 Da), which retains the antibody–adduct complex. Filters were washed five times with water and the remnants collected by inversion of the filter and short centrifugation (10 000 r.p.m. for 5 min). Proteins were precipitated by the addition of 500 µl cold ethanol and the supernatants were dried and stored at –80°C. Internal standard (800 attomol {varepsilon}dA) and 4 µl kinase buffer were added to the dried enriched samples. After addition of 2 µl of [{gamma}-32P]ATP (20 µCi), 10 U T4-PNK were added and the mixture was incubated for 2 h at 37°C. Another 10 U T4-PNK were added after 1 h of incubation. Polyethyleneimine (PEI) minicolumns were prepared by filling 2.5 mg PEI into a 200 µl pipet-tip closed with a small frit. The labeled samples were pipetted on top of the PEI and centrifuged for 5 min at 10 000 r.p.m. at 4°C. The labeled adducts were eluted with 5x25 µl 1 M acetic acid, whereas excess ATP bound to the PEI. The eluent was injected into a C18-HPLC with online detection of radioactivity. The mobile phase consisted of 100 mM ammonium formate pH 7.5 with 5% MeOH during the first 12 min at a flow rate of 1 ml/min. Then, the methanol concentration was increased to 20% over an 8 min time period (from 12 to 20 min) and further increased to 50% over a period of 10 min (from 20 to 30 min). The internal standard eluted at 17 min and O4etT-5' 32P-monophosphate at 22 min, whereas residual normal nucleotides eluted before 12 min as shown in Figure 1AGo (sample) and D (standard).



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Fig. 1. Typical DNA adduct profiles for respectively O4-ethylthymidine (A), etheno–DNA adducts (B) and aromatic–DNA adducts (C). (DF) Represent the corresponding standards.

 
1,N6-ethenodeoxyadenosine ({varepsilon}dA), 3,N4-ethenodeoxycytidine ({varepsilon}dC). {varepsilon}dA and {varepsilon}dC were analyzed in DNA by immunoaffinity/32P-postlabeling (15). In brief, 25 µg of DNA was hydrolyzed to nucleotide 3'-monophosphates using micrococcal nuclease and spleen phosphodiesterase. Normal nucleotides were quantified by high-performance liquid chromatography of part of the digest and the adducts were enriched on immunoaffinity columns prepared from the monoclonal antibodies EM-A-1 ({varepsilon}dA) and EM-C-1 ({varepsilon}dC). The antibodies used in this study were provided by M.Rajewsky (Institute of Cell Biology, University of Essen, Essen, Germany); their characteristics have been reported previously. The adducts and the internal standard deoxyuridine 3'-monophosphate were labeled with [{gamma}-32P]ATP (>5000 Ci/mmol) and T4 polynucleotide kinase (Amersham Buchler, Braunschweig, Germany and Pharmacia Biotech, Freiburg, Germany, respectively). The adducts were resolved on polyethyleneimine-TLC plates using two-directional chromatography [D1 = 1 M acetic acid (pH 3.5), D2 = saturated ammonium sulfate (pH 3.5)]. After autoradiography, the adduct spots and the internal standard were marked, cut, and the radioactivity was measured in a liquid scintillation counter. The absolute adduct levels were quantified using standards, and the relative adduct level per parent nucleotides was determined with the amount of deoxycytidine (dC) and deoxyadenosine (dA) obtained from high-performance liquid chromatography analysis of normal nucleotides obtained from the DNA digest. Typical chromatograms are shown in Figure 1BGo (sample) and E (standard).

Aromatic–DNA adducts.
DNA (~10 µg) was digested into nucleotide monophosphates and treated with nuclease P1 for 40 min at 37°C (16). Then, nucleotides were 5'-labeled with 32P using T4-polynucleotide kinase (5.0 U) for 30 min at 37°C. Radiolabeled adduct nucleotide biphosphates were separated by chromatography on PEI-cellulose sheets (Macherey Nagel, Germany). The following solvent systems were used: 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 M urea pH 8.0; D4, 1.7 M NaH2PO4 pH 6.0. In each experiment, three standards of [3H]BPDE modified DNA with known modification levels (1/107, 108, 109 nucleotides) were run in parallel for quantification purposes. Quantification was performed by using phosphor imaging technology (Molecular Dynamics, Sunnyvale). Detection limits of <0.1 adducts/108 nucleotides were obtained for [3H]BPDE–DNA. A typical chromatogram is shown in Figure 1CGo (sample) and F (standard). Adducts were quantified as total aromatic–DNA adducts (total radioactivity) and as putative BPDE–DNA adducts (radioactivity at chromatographic location of BPDE–DNA).

Statistics
Data are presented as means (±SD). Non-parametric tests were used to assess statistical significances; the Mann–Whitney U-test was used to assess differences between DNA adduct levels in smokers and non-smokers and the Friedman test for paired samples was used to assess differences between the levels of various DNA adduct types. Adduct levels of samples in which no adducts were detectable, were arbitrarily set on half of the detection limit. Relationships between DNA adduct levels were determined by simple regression analysis. P < 0.05 was considered statistically significant.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
O4etT in human lung
O4etT reached detectable levels in 10 out of 13 smokers, but only in three out of 11 non-smokers (limit of detection is ~2 adducts/108 thymidines). O4etT levels were ~2.4-fold higher in smoking lung cancer patients as compared with non-smoking subjects (3.8 ± 2.1 O4etT/108 thymidines versus 1.6 ± 1.3, respectively, P = 0.003, Table IIGo). Overall O4etT levels ranged from <2.0 to 8.6 adducts/108 normal thymidines. Levels of O4etT in human lung were not found to be related to age, gender, histological tumor-type or tumor-stage.


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Table II. Relative levels of PAH–DNA adducts, O4-ethylthymidine and etheno–DNA adducts in human lung, expressed either as adducts per 108 parent nucleotides or adducts per 108 total nucleotides (mean ± SD and range)
 
PAH–DNA adducts
Aromatic–DNA adduct profiles showed diagonal radioactive zones (DRZ) in all smokers except one, which is found to be typical for exposure to complex chemical mixtures. Faint DRZs were found in non-tumorous tissue of five out of 11 non-smoking subjects. Total aromatic–DNA adduct levels were significantly higher in smoking lung cancer patients as compared with non-smoking patients (11.2 ± 7.8 adducts/108 nucleotides and 2.2 ± 2.2, respectively, P = 0.001, Table IIGo). A positive correlation was observed between aromatic–DNA adducts and O4etT, indicating that both were formed from the same source of exposure, i.e. cigarette smoke (r = 0.66, P < 0.01, Figure 2Go, smokers and non-smokers combined). This relationship was also found after exclusion of non-smokers from the analysis (r = 0.54, P = 0.05). To get an impression of the BPDE–DNA adduct level, DNA adducts that migrated at the position of BPDE–DNA were additionally quantified. Putative BPDE–DNA adduct levels were 1.5 ± 1.0/108 nucleotides in smoking subjects and 0.2 ± 0.2 in non-smokers (P < 0.001), which is most probably an overestimation of the actual BPDE–DNA adduct level, because other adducts may co-migrate during TLC.



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Fig. 2. Correlation between O4-ethylthymidine and aromatic–DNA adducts in human lung of smokers and non-smokers (R = 0.66, P < 0.01). (Insert) Correlation between O4-ethylthymidine and putative BPDE–DNA adducts (R = 0.64, P < 0.01).

 
Etheno–DNA adducts in human lung
{varepsilon}dA and {varepsilon}dC levels were not influenced by smoking behavior (Table IIGo), but were highly interrelated (R = 0.76, P < 0.001), suggesting that both adduct types were formed by similar pathways. Median {varepsilon}dA levels were 9.2 adducts/109 parent nucleotides (range 2.6–146) and 9.4 (range 2.4–56.5) in non-smokers and smokers, respectively. Median {varepsilon}dC levels were 19.7/109 parent nucleotides (range 1.6–395.5) and 9.8 (range 5.8–80.9) in non-smokers and smokers, respectively. Large inter-individual variations were observed; {varepsilon}dA levels ranged from 1.8 to 146.0 adducts/109 parent nucleotides (80-fold difference), whereas {varepsilon}dC levels ranged from 1.6 to 395.5 adducts/109 parent nucleotides (250-fold difference). Interestingly, these inter-individual variations were much higher in non-smokers (56- and 250-fold for {varepsilon}dA and {varepsilon}dC, respectively) than in smokers (31- and 19-fold for {varepsilon}dA and {varepsilon}dC, respectively). Etheno–DNA adduct levels were not related to age, gender, histological tumor-type or tumor-stage.

Adduct abundancy
For reasons of comparison, the relative adduct levels of all adduct types were calculated as adducts per total nucleotides (Table IIGo). O4etT levels were 0.5 ± 0.4 adducts/108 nucleotides and 1.2 ± 0.6 for non-smokers and smokers, respectively. Quantification of aromatic–DNA adducts that migrated at the position of BPDE–DNA in smokers resulted in mean levels of 1.5 ± 1.0 adducts/108 nucleotides (range <0.1–2.6), which was not statistically different as compared with the levels of O4etT in the same individuals (range 0.6–2.5). When {varepsilon}-DNA adduct levels were also expressed as adducts per 108 total nucleotides, {varepsilon}dA levels were found to be 1.0 ± 1.6 in non-smokers (range 0.1–4.3) and 0.5 ± 0.5 in smokers (range 0.1–1.5), which was different from the levels of O4etT and putative BPDE–DNA in smokers, but not in non-smokers. For {varepsilon}dC similar results were found (Table IIGo). In all smokers and non-smokers, BPDE–DNA like adducts, O4etT and etheno–DNA adducts were detected at similar levels in lung DNA (Table IIGo).


    Discussion
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 Materials and methods
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Several studies indicated that the formation of PAH–DNA adducts, particularly BPDE–DNA, in human lung is involved in tobacco smoke-induced lung carcinogenesis (3,4). In the present study total aromatic–DNA adducts and putative BPDE–DNA adducts were found to be higher in smokers than in non-smokers by 32P-postlabeling. In other studies on PAH–DNA adducts in human lung, similar results were obtained by 32P-postlabeling (5,6,17,18), but the quantitative aspects should be regarded with some caution, because of uncertainties in the efficiency of 32P-labeling, recovery and identification of adducts. Nonetheless, putative BPDE–DNA adduct levels as determined in the present study are comparable with those levels reported for human lung using an HPLC-fluorescence detection methodology, with which specific BPDE–DNA adducts can be detected and quantified (19,20).

Our newly developed methodology to measure O4etT in small quantities of DNA enabled the analysis of human lung samples. O4etT levels were significantly increased in smokers as compared with non-smokers and the levels correlated with aromatic–DNA adduct levels, indicating that both originate from cigarette smoke as the main source of exposure. Although active removal of O4etT from human DNA was reported (21), the repair mechanisms have not yet been elucidated. The current evidence suggests that O4etT is only poorly repaired by O6-alkylguanine-DNA-alkyltransferase (12). Nucleotide excision repair seems to be involved (12,22), which is in agreement with the observed close relationship between O4etT and PAH–DNA adducts in the present study, as bulky PAH–DNA adducts are also repaired by nucleotide excision repair.

Direct comparison of O4etT and BPDE–DNA adducts normalized to total nucleotides, showed that both adducts were equally abundant in lung DNA of smokers (Table IIGo), and therefore the highly promutagenic O4etT may contribute equally to the increased genomic instability and increased cancer risk in smokers. Ethylating compounds are rare as environmental contaminants, but they were undoubtedly found in cigarette smoke (such as NDEA, 0.1–28 ng/cigarette; 23). Increased urinary excretion of N-ethylated adenine has been reported for smokers as compared with non-smokers (911) and the measurement of O4etT was therefore expected to offer a highly specific DNA modification to assess exposure to tobacco smoke. Until now, other types of carcinogen–DNA adducts have been used for this purpose, but due to their ubiquitous presence in the environment it could not be established from what source the carcinogens originate. For example, PAH occur in cigarette smoke, but can also be found in large quantities in food products. Oral and inhalation exposure to these compounds might both lead to detectable DNA adducts in lung tissue (24), and are thus thought to be unspecific for cigarette smoke. In the present study, we also have detected O4etT and PAH–DNA adducts in lung DNA of non-smokers. The source of these background levels could be the environment, environmental tobacco smoke or both. We could neither confirm nor exclude a role of ETS, due to limited exposure data of these patients. NDEA levels were found to be up to 40 times higher in side stream cigarette smoke as compared with main stream smoke (7). Also, in studies on the urinary excretion of ethylated DNA bases (911) and in a study on the ethylation of hemoglobin (25), background levels were observed in non-smoking subjects. Recently, the ethylation and methylation of N-terminal valine of hemoglobin was investigated (25) and a difference was found between smokers and non-smokers for ethyl-valine but not for methyl-valine. Similar results were reported for O4-ethyl- and O4-methyl-thymidine in human lung DNA (26). These observations indicate that measuring DNA ethylation is more specific for cigarette smoke exposure than methylation.

Surprisingly, no effect of smoking was found on the formation of etheno–DNA adducts, which is not in line with previous studies that found slightly elevated 8-hydroxy-deoxyguanosine levels (~1.5-fold) as marker for oxidative stress in cigarette smokers (27). Most subjects in the present study reported to have stopped smoking at least 1 week before surgery. Therefore, effective DNA repair of etheno–DNA adducts may explain why we did not see a difference between smokers and non-smokers. Our data also indicate that lung tissue is well protected against LPO induced by exogenous radicals, because the levels of background etheno–DNA adducts as reported in this study, were lower as compared with the levels reported for other human tissues, such as colon-epithelium, liver, pancreas, oesophagus and leucocytes (14). Still, in some individuals high levels of etheno–DNA adducts were found, which could be due to increased formation or high persistence (e.g. poor repair) of the adducts. Individuals may significantly differ in their effectivity for removal of {varepsilon}dA and {varepsilon}dC, because the {varepsilon}dC:{varepsilon}dA ratio ranged from 0.1 to 7.5 (data not shown); {varepsilon}dA is repaired by 3-methyladenine DNA glycosylase, whereas {varepsilon}dC is repaired by a mismatch specific thymidine-DNA glycosylase (28). Highest etheno–DNA adduct levels were found in non-smoking lung cancer subjects with low levels of PAH–DNA adducts or O4etT, suggesting that {varepsilon}-DNA adducts may be involved in the onset or progression of lung cancer in non-smoking subjects. However, more studies are necessary to find biological reasons and implications of the large inter-individual differences in the levels of etheno-bridged DNA bases.

Table IIIGo shows a brief summary of major DNA adduct types detected in human lung DNA. Studies reporting adduct levels in both smokers and non-smokers were included. Though higher adduct levels were reported for 8-hydroxy-dG in human lung, only DNA adducts by ethylating compounds (i.e. O4etT and O6-ethyl-dG), bulky PAH–DNA adducts, BPDE–DNA and the HPB-releasing adducts were found to be >2-fold increased (range 2–14-fold) in smokers as compared with non-smokers, suggesting that these adduct types are derived from tobacco smoke carcinogens and can be used as biomarkers for exposure to cigarette smoke. Overall cumulative levels of these adducts were 48.7 and 20.1 base modifications/108 nucleotides for smokers and non-smokers, respectively. We conclude that in addition to DNA adducts derived from PAH and TSNA (29), miscoding O4etT lesions are formed by cigarette smoke that contribute to the increased genomic instability and lung cancer risk in smokers.


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Table III. Literature data and results from this study on levels of various DNA adduct types (number of adducts per 108 nucleotidesa) in non-tumorous lung tissue of smokers and non-smokers
 


    Notes
 
* This article is dedicated to Harald zur Hausen on the occasion of his retirement as head of the German Cancer Research Center with gratitude and appreciation for 20 years of leadership. Back

4 To whom correspondence should be addressed Email: j.nair{at}dkfz.de Back


    Acknowledgments
 
Roger Godschalk was recipient of a EU-Marie Curie fellowship. The authors would like to thank Marcel van Herwijnen (Maastricht University, Maastricht, The Netherlands) and Christel Ditrich (German Cancer Research Center, Heidelberg, Germany) for their technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 6, 2002; revised October 7, 2002; accepted October 8, 2002.