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Modified Immunoenriched 32P-HPLC Assay for the Detection of O4-Ethylthymidine in Human Biomonitoring Studies Roger Godschalk,† Jagadeesan Nair,*,† Hans-Christian Kliem,‡ Manfred Wiessler,‡ Guy Bouvier,§ and Helmut Bartsch† Divisions of Toxicology and Cancer Risk Factors and Molecular Toxicology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Received November 7, 2001
Increased excretion of ethylated DNA bases has been reported in the urine of cigarette smokers. To study DNA ethylation in the target organs of smokers, an immunoenriched 32Ppostlabeling assay for O4-ethylthymidine (O4-etT) was developed. O4-etT-3′-monophosphate (O4-etT-3′P) was synthesized, purified, and characterized by LC-MS, ESI-MS, and NMR. DNA was enzymatically digested to 2′-deoxynucleoside-3′-monophosphate followed by immunoprecipitation of O4-etT-3′P using specific monoclonal antibodies. The immunoconjugate was washed by filtration, and O4-etT-3′P was recovered by ethanol treatment. The enriched O4-etT-3′P was labeled with [γ-32P]ATP in the presence of T4-polynucleotide kinase at pH 6.8 to yield its 5′labeled monophosphate and was subsequently resolved on RP-HPLC and detected with online detection of radioactivity. Adduct recovery was >80%, and the detection limit was approximately 500 amol. To further validate the method, O4-etT levels were determined in calf thymus DNA treated with N-ethyl-N-nitrosourea, and a dose-dependent formation of O4-etT was observed. Furthermore, O4-etT was found to be present in the cells obtained from the lower respiratory tract by sputum induction of two out of four smokers but not in three nonsmokers. O4-etT is a poorly repaired promutagenic DNA lesion; thus, it could be of potential use for biomonitoring smoking-related DNA damage. Our improved assay was found to be sufficiently sensitive and specific to detect O4-etT in surrogate cells from cigarette smoke exposed humans.
Introduction Alkylating agents react extensively with DNA to produce several types of promutagenic structural base modifications (1), which represent the biologically effective dose that can be used in exposure and possibly in risk assessment (2). Tobacco smoke contains many alkylating compounds, and their mutagenic potential mainly depends on the position of binding within the four DNA bases (3, 4). O-Substitution by alkylating agents results in relevant DNA adducts in terms of mutagenesis and carcinogenesis, including O6-alkylguanine and O4-alkylthymine. Animal studies indicated that, after exposure to ethylating compounds, O4-ethylthymidine (O4-etT)1 is initially formed in very low levels but is poorly repaired (5) and thus accumulates to biologically relevant levels. Also, cigarette smoke contains hitherto uncharacterized ethylating agents, because increased levels of N-ethylated DNA bases have been reported in the urine of smokers as compared to nonsmokers (6-8). Highly sensitive methods for the detection of O4-etT are required for human biomonitoring and to further investigate the role * Corresponding author. E-mail:
[email protected]. Phone: +49 6221 42 3306/3301. Fax:+49 6221 42 3359. † Division of Toxicology and Cancer Risk Factors, German Cancer Research Center. ‡ Division of Molecular Toxicology, German Cancer Research Center. § Current address: Aventis Crop Science, Regulatory Toxicology Insecticide/Fungicide, 355 rue Dostoı¨evski, BP 15306903 Sophia Antipolis, France. 1 Abbreviations: O4-etT, O4-ethylthymidine; dA, 1,N6-ethenodeoxyadenosine; I.S., internal standard; ENU, N-ethyl-N-nitrosourea.
of O4-etT in tobacco smoke-induced carcinogenesis, whereby the amount of DNA available from target organs is often limited. To date, the 32P-postlabeling assay is widely applied to measure structurally diverse DNA modifications, including alkylation products (9, 10). A 32Ppostlabeling method was previously developed to assess O4-etT levels in human samples (10), but this assay required relatively large amounts of DNA (up to 100 µg) to reach a reasonable limit of detection and was timeconsuming. Therefore, we developed a modified immunoenriched 32P-postlabeling procedure with which subfemtomole levels of O4-etT could be reliably detected. This method was further validated by applying it to DNA treated with the direct-acting ethylating carcinogen N-ethyl-N-nitrosourea (ENU) and to DNA obtained from cells of the lower respiratory tract of smokers and nonsmokers.
Experimental Procedures Materials and Equipment. (1) Materials. 3′-Monophosphate of O4-etT was synthesized using 5′-dimethoxytrityl-3′phosporamidite thymidine (Amersham/Pharmacia Biotech, Freiburg, Germany) (11). Calf thymus DNA (sodium salt) and micrococcal endonuclease were purchased from Sigma-Aldrich (Taufkirchen, Germany). Spleen phosphodiesterase was obtained from Worthington Biochemical Corp. (Lakewood, NJ) and T4-polynucleotide kinase (T4-PNK) from Amersham/Pharmacia Biotech (Freiburg, Germany). [γ-32P]ATP with a specific activity of >220 TBq/mmol was purchased from Hartmann Analytic (Braunschweig, Germany). (Caution: [γ-32P]ATP is a hazardous radioactive compound and should only be handled with
10.1021/tx015582s CCC: $22.00 © 2002 American Chemical Society Published on Web 02/22/2002
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Figure 1. Scheme for the synthesis of O4-ethylthymidine-3′monophosphate (4): DMTr, 4,4′-dimethoxytrityl. sufficient protection and shielding.) Polyethyleneimine (PEI) was obtained from J.T. Baker, Inc. (Phillipsburg, NJ). Antibody ER01 (12), which specifically recognizes O4-etT-3′- and 5′-monophosphates, was provided by Dr. P. Lorenz (Uni-Klinikum, University of Essen, Germany) and was purified before use by an antibody purification kit (Immunopure (A/G) IgG purification kit, Pierce, Rockford, IL). Antibodies were subsequently concentrated on centrifugation filters (MY-50, Millipore, Bedford, MA). (2) Equipment. An HPLC-model 1090 from Hewlett-Packard (Waldbronn, Germany) with a 250 × 4 mm C18 column (Bischoff, Leonberg, Germany) was connected to an EG&G Berthold (Bad Wildbad, Germany) radioactivity detector with cell-model Z200-4. Synthesis of O4-etT-3′-Monophosphate Standard. (1) 5′Dimethoxytritylthymidine-3′-(biscyanoethyl)monophosphate 2 (see Figure 1). The solution of commercially available thymidine-3′-phosphite 1 (500 mg, 0.67 mmol) in 10 mL of acetonitrile was incubated with 0.5 mL of hydroxypropionitrile and 2.3 mL of tetrazole (1 mM solution in acetonitrile) at ambient temperature. After 90 min, 10 mL of a solution of 3% cumene-hydroperoxide in acetonitrile was added to oxidize the phosphite diester to phosphate 2. The reaction was stopped after 2 h by adding 3 mL of ethanol, then diluted with 50 mL of chloroform, and washed with a concentrated aqueous solution of NaHCO3 (2 × 30 mL) and water (2 × 30 mL). The organic layer was separated, dried with Na2SO4, and filtered, and the solvent was removed in a vacuum. The resulting product was purified by flash chromotography (silica gel, chloroform/ methanol (95/5, v/v)) to yield 2 (187 mg, 38%). ESI-MS (m/z): 753.0 ([M + Na+], 100%). (2) O4-Ethyl-5′-dimethoxytritylthymidine-3′-(biscyanoethyl)monophosphate 3. A freshly prepared solution of diazoethane (approximately 8 mmol) in diethyl ether (10 mL) was poured into a solution of 2 (185 mg, 0.25 mmol) in 5 mL of methanol to form the O4-ethylated adduct 3. (Caution: Diazoethane is a hazardous alkylating compound and should be handled carefully.) After 30 min, the solvents were removed in a vacuum, and the residue was purified by flash chromatography (chloroform/ethanol (98/2, v/v)) to yield compound 3 (26 mg, 13%). ESI-MS (m/z): 781.2 ([M + Na+], 40%), 1539.6 ([2M + Na+], 100%). (3) O4-Ethylthymidine-3′-monophosphate 4. For 5′-deprotection, 3 (25 mg, 33 mmol) was dissolved in 2 mL of dry nitromethane, and 2 mL of a saturated solution of ZnBr2 in nitromethane was added. The color of the reaction solution turned bright orange. The reaction was quenched with 40 mL
Godschalk et al. of an aqueous solution of ammonium acetate (1 M) and extracted with 80 mL of chloroform. The organic solvent was dried over Na2SO4, filtered, and removed in a vacuum. For deprotection of the 3′-phosphate group, the residue was incubated with 10 mL of ammonia for 3 h. Ammonia was removed in vacuo, and the solution was then lyophilized. Reversed-phase chromatography (0-50% acetonitrile/water in 30 min) yielded pure 4 (4.1 mg, 35%). ESI-MS (m/z): 351.2 ([M + H+], 45%), 373.9 ([M + Na+], 17%), 701.1 ([2M + H+], 100%), 723.3 ([2M + Na+], 30%); 349.1 ([M - H]-, 100%). 31P NMR (101 MHz, D2O, δ): 0.228 (brs, P). 13C NMR (62 MHz, D2O, δ): 174.3 (C-4), 160.5 (C-2), 143.2 (C-6), 110.5 (C-5), 89.2 (C-4′), 88.9 (C-1′), 77.1 (C-3′), 67.1 (C-5′), 63.9 (O4-C), 441.4 (C-2′), 16.2 (CH3), 14.1 (CH3). 1H NMR (250 MHz, D2O, δ): 7.88 (q, 1H, H-6), 6.30 (dd, 1H, H-1′), 4.40 (q, 2H, O4-CH2), 4.20 (m, 1H, H-4′), 3.88 (dd, 1H, H-5′a), 3.80 (dd, 1H, H-5′b), 2.64 (ddd, 1H, H-2′a), 2.35 (ddd, 1H, H-2′b), 1.98 (d, 3H, C5-CH3), 1.38 (t, 3H, O4-C-CH3) (J1′,2′a ) J1′,2′b ) 6.5 Hz, J2′a,2′b ) 13.9 Hz, J2′a,3′ ) 4.2 Hz, J2′b,3′ ) 6.7 Hz, J4′,5′a ) 3.3 Hz, J4′,5′b ) 4.9 Hz, J5′a,5′b ) 12.5 Hz, J5,CH3 ) 1.1 Hz, JCH2,CH3 ) 7.1 Hz; H-3 is covered by the signal of D2O; q ) quadruplet). LC-MS: the mole peak [M - H]- and a dimer peak [2M - H]were observed at m/z: 349 and 699, respectively, with a major fragment ion at m/z: 195 representing the base-free 2′-deoxyribose-3′-monophosphate. In Vitro Treatment of Calf Thymus DNA with ENU. Calf thymus DNA was dissolved in water at a concentration of 0.4 mg/mL, and N-ethyl-N-nitrosourea (ENU) dissolved in DMSO was added to reach final concentrations of 20, 2.0, 0.2, 0.02, and 0.002 µmol of ENU/incubation of 200 µL (Caution: N-ethylN-nitrosourea is a hazardous carcinogenic compound and should be handled carefully). After incubation for 1 h at 37 °C, DNA was precipitated by the addition of 1/30 volume of sodium acetate (3 M, pH 5.2) and 2 volumes of ice-cold ethanol. DNA was washed twice with 70% ethanol, dried in vacuo, and redissolved in distilled water at a concentration of 1 µg/µL. Sputum Induction and DNA Isolation from Smokers and Nonsmokers. After clearance by the medical ethical committee of the Maastricht University (The Netherlands), four smokers and three nonsmokers volunteered to undergo sputum induction to obtain cells from the lower respiratory tract (mostly bronchoalveolar macrophages). Sputum induction was performed by inhalation of ultrasonically nebulized 4.5% saline delivered from an Ultra-Neb 2000 (De Vilbiss, Somerset, PA). The subjects all produced expectorate into a 50-mL tube, and the induced sputum was processed within 2.5 h of sampling, as previously described by Besarati-Nia et al. (13). Cells were lysed with 0.5% SDS in 100 mM NaCl, 20 mM EDTA, and 50 mM Tris (pH 8.0) at 37 °C overnight. The resulting suspension was treated with RNase for 3 h at 37 °C, followed by treatment with proteinase K. DNA was isolated by extraction with phenol/ chloroform/isoamyl alcohol (25/24/1, by vol) and chloroform/ isoamyl alcohol (24/1), respectively, and precipitated with 2 volumes of 100% cold ethanol and 1/30 volume of 3 M sodium acetate (pH 5.3). Precipitated DNA was rinsed with 70% ethanol and dissolved in 2 mM Tris (pH 7.4). Purity and concentration of the DNA were determined spectrophotometrically by absorbance at 230, 260, and 280 nm, and the concentration was adjusted to 2 mg/mL. Digestion of DNA and Immunoenrichment. DNA dried in vacuo (25 µg) was reconstituted in 27.5 µL of a Tris-buffer (80 mM Tris and 20 mM CaCl2 (pH 6.8)) and subsequently digested to 3′-monophosphate nucleosides by the addition of 12.5 µL of micrococcal endonuclease (0.2 units/µL) and 10 µL of spleen phosphodiesterase (0.025 units/µL) followed by incubation at 37 °C for 4 h (14). Immunoprecipitation was performed according to a modified procedure of Kang et al. (10). Fifty microliters of immunobuffer (10 mM Tris (pH 7.5), 140 mM NaCl, 3 mM NaN3, 1% BSA, and 0.1% rabbit IgG) and 50 µL of purified monoclonal antibody were added and incubated for 1 h at room temperature with continuous mixing. Saturated ammonium sulfate was added (1.3 mL) and placed on ice for 30
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Figure 3. Mean recovery of O4-etT ((SD) using different concentrations of the specific antibody ER-01 in three independent experiments in which 25 µg of calf thymus DNA was spiked with 1 fmol of O4-etT. Figure 2. Profiles for (A) O4-ethylthymidine by HPLC with online detection of radioactivity: (I) calf thymus DNA exposed to ENU, (II) DNA of a smoker’s induced sputum, and (III) DNA spiked with O4-etT standard. (B) Analysis of normal nucleotides by HPLC-UV: I.S., internal standard. min. The antibody-adduct interaction product was collected by centrifugation and was redissolved in 400 µL of water (the supernatant was used for the quantitation of normals; see the next section). This solution was transferred into a microcon centrifugation filter (MY-50; molecular weight cutoff, 50 000 Da), which retains the antibody-adduct complex but not residual normal nucleotides and excess of ammonium salt. Filters were washed 5 times with water (450 µL), and the retentates were recollected by inversion of the filter and short centrifugation (10 000 rpm for 5 min). Proteins were precipitated by the addition of 500 µL of cold ethanol, and the supernatants were dried and stored at -80 °C. Recovery of O4-etT was determined by using purified O4-etT-5′-32P-monophosphate (for labeling conditions, see the following discussion) and by the comparison of peak areas obtained from spiked calf thymus DNA with those of adduct standards. Quantitation of Amount of DNA in the Analysis. After precipitation of antibodies with ammonium sulfate, the supernatant contained all unbound material, including normal nucleotides. To determine the amount of DNA in the analysis, 10 µL was analyzed by reversed-phase HPLC, isocratically eluted with 100 mM ammonium formate (pH 7.5). The normals were detected by UV absorption at 260 nm and quantitated by comparison with standard solutions with known amounts of nucleotides (200 pmol of each nucleotide). A typical chromatogram is shown in Figure 2B. Labeling and HPLC Online Analysis of O4-etT. Internal standard (800 attomol 1,N6-ethenodeoxyadenosine, dA; see ref 14 for details) and 4 µL of a kinase buffer (125 mM Tris-HCl, 25 mM MgCl2, and 25 mM DTT (pH 6.8)) were added to the dried enriched samples. After the addition of 2 µL of [γ-32P]ATP (20 µCi), 10 units of T4-PNK were added, and the mixture was incubated for 2 h at 37 °C. Another 10 units of T4-PNK were added after 1 h of incubation. Polyethyleneimine (PEI) minicolumns were prepared by filling 2.5 mg of PEI into a 200 µL pipet tip closed with a small fritt. The labeled samples were pipetted on top of the PEI and centrifuged for 5 min at 10 000 rpm at 4 °C. The labeled adducts were eluted with 5 × 25 µL of 1 M acetic acid, whereas excess ATP bound to 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 O4-etT5′32P at 22 min, whereas residual normal nucleotides eluted before 12 min (Figure 2A). In the case of high background radioactivity by tailing of residual normal nucleotides, appropriate fractions were collected, dried, and reinjected.
Figure 4. Linear relationship between the peak area of O4etT and the peak area of a fixed amount of internal standard (800 amol of 1,N6-ethenodeoxyadenosine, dA).
Results Analysis of O4-Ethylthymidine. Numerous attempts to prepare immunoaffinity columns for O4-etT were unsuccessful. Therefore, O4-etT was enriched by immunoprecipitation and subsequent desalting of the adductantibody conjugate on membrane filters, which enabled the analysis of O4-etT-3′P levels without inhibition of labeling due to excessively high salt concentrations. Typical HPLC chromatograms for the detection of O4etT are shown in Figure 2A. Adduct levels were related to the amount of normal thymidine present in each sample as determined by HPLC-UV (Figure 2B). The recovery of adducts depended on the antibody concentration; the use of unconcentrated antibodies (13 µg/mL) resulted in a maximal recovery of O4-etT of less than 50%, whereas 5-fold concentrated antibodies (65 µg/mL) showed a reproducible recovery of >80% (Figure 3), and this antibody concentration was therefore used for the rest of the experiments. 1,N6-Ethenodeoxyadenosine (dA; 800 amol) was added to all samples as an internal standard for quantitation purposes. A linear relationship between the amount of O4-etT and dA was observed (Figure 4). The labeling efficiency of O4-etT was found to be 70%, but in the presence of internal standard (800 amol) and normal nucleotides (10 fmol of each), the labeling efficiency decreased to ca. 20%. Increasing the amount of ATP or labeling by the method as originally described by Kang et al. (10) did not improve the labeling efficiency, which appears to be a major limitation for the detection of O4etT. Optimal conditions were obtained by labeling at pH 6.8 and two additions of 10 units of T4-PNK. DNA Adduct Levels in Calf Thymus DNA Treated with ENU. To further validate this method, calf thymus DNA was treated with increasing concentrations of ENU
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Figure 5. Sigmoidal dose-response relationship of O4-etT in ENU treated calf thymus DNA.
for 60 min and analyzed for the presence of O4-etT in triplicate (sample I in Figure 2A). A clear dose-response was observed (Figure 5) with adduct levels ranging from 44.5 ( 12.4 (mean ( SD) to 2913.8 ( 428.1 O4-etT/108 normal thymidines. The overall assay variation was found to be approximately 20%. DNA Adduct Formation in Induced Sputum from Smokers and Nonsmokers. To test the applicability of this newly developed assay for human samples, cells from the lower respiratory tract were obtained by sputum induction and analyzed for the presence of O4-etT. O4etT was found in low levels in two out of four smokers (sample II in Figure 2A) and in none of the nonsmokers. O4-etT levels in the two smokers with detectable levels were found to be 2.4 and 4.0 adducts/108 thymidines. In all other samples, adduct levels were e2.0 adducts/108 thymidines (detection limit).
Discussion O4-etT-3′P was synthesized and characterized by various spectrometric data, which enabled us to systematically investigate stability, recovery, and labeling efficiency of this biologically relevant DNA-alkylation product. Among about a dozen different alkylation products induced in genomic DNA by carcinogenic N-nitroso compounds, O4-etT can be considered as one of the major promutational lesions causing TA f CG transitions (15). Although O4-etT is initially formed at far lower amounts in cellular DNA than are other alkylation products, it accumulates because of its inefficient repair and may, thus, play an important role in human tobacco-induced cancers (5, 16). The possible involvement of ethylating agents in cigarette smoke has been demonstrated in humans by studies on urinary excreted N-3-ethylated adenine (6-8). To further investigate the role of O4-etT in tobacco smoke-induced malignancies, a sensitive and specific assay with which subfemtomole levels of O4-etT can be detected was developed. The assay described in this study is capable of detecting 500 amol of O4-etT, which is ca. 2-fold more sensitive than the methodology developed previously (10). It thus offers the possibility to use smaller amounts of DNA (approximately 25 µg) to reach a similar relative detection limit of 1-2 adducts/ 108 parent nucleotides. Furthermore, this new enrichment procedure for DNA adducts is quick and, in principle, applicable to all types of DNA adducts for which antibodies are available. In this modified method, repeated washings of the adduct-antibody conjugate on membrane filters effectively removed excessive ammonium salts (because of the precipitation with ammonium sulfate) and enabled the subsequent analysis of
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O4-etT-3′P levels without inhibition of 5′-labeling. The specificity of antibodies is combined with the high sensitivity of 32P-postlabeling, and this method can also be used in conjunction with other enrichment procedures, for example, HPLC preseparation as described previously for O4-etT (10). Over 15 years ago, an immunoslot-blot (ISB) methodology for the detection of O4-etT had been described (17). Although this ISB method has a higher absolute sensitivity for O4-etT (100 amol) as compared to our method (500 amol), it may not reach the sensitivity required for human DNA samples because of the limited amount of DNA that can be used in the assay (e3 µg). The lowest molar ratio which can be detected by ISB was reported to be 100 amol of O4-etT in 3 µg of DNA (17) (i.e., 4 adducts/108 normal thymidines). Thus, the major advantage of our method is that more DNA can be used, which permits the measurement of relative DNA adduct levels as low as 2-4 adducts/108 thymidines, as observed in human sputum samples. Although the active removal of O4-etT from human DNA was reported (18), the repair mechanisms have not yet been elucidated. It is suggested that nucleotide excision repair is involved (19), and the current evidence suggests that O4-etT is only poorly repaired by O6alkylguanine-DNA alkyltransferase (5). O4-etT was found to be highly persistent in animal tissues (16). Thus, even small amounts of O4-etT in genomic DNA may have biological consequences, and a method to quantify this type of adduct requires high sensitivity. The relative detection limit of ∼2 adducts/108 normal thymidines (500 amol of O4-etT in 25 µg of DNA) was found to be sufficiently low to detect O4-etT in human lung samples of smokers, in which the mean adduct levels were found to be approximately 4-8 adducts/108 thymidines (20). Exposure of calf thymus DNA to increasing concentrations of ENU resulted in a dose-dependent formation of O4-etT, with an overall assay variation of less than 20%. Ethylating compounds are rarely present as environmental contaminants. Therefore, the measurement of O4etT may offer a highly specific DNA modification to study exposure to tobacco smoke, although the nature of the DNA-reactive agent(s) remains to be determined. Until now, other types of tobacco-derived carcinogen-DNA adducts have been used for this purpose, but in some cases, because of their ubiquitous presence in the environment, it could not be unequivocally established from what source these carcinogens originate. For example, polycyclic aromatic hydrocarbons occur in cigarette smoke, but can also be found in large quantities as food byproducts. Oral and inhalatory exposure to these hydrocarbons may both lead to detectable DNA adducts (21) and are thus unspecific indicators for cigarette smoke exposure. In addition to pyridyloxobutyl-DNA adducts, which provided a promising approach for human dosimetry of major tobacco specific N-nitrosamines (22), O4-etT may represent another useful specific biomarker for cumulative exposure to cigarette smoke, because (i) urinary excretion of ethylated DNA-bases proved to be independent from diet (6) and (ii) in our feasibility study, O4-etT was detected in human cells obtained from the lower respiratory tract of smokers but not in those of nonsmokers. Moreover, smoking related O4-etT was previously detected in human lung tissue, with adduct levels being 2-fold higher in smokers than in ex-smokers, whereas, in the same samples, mean levels of O6-methylguanine and O4-methylthymine did not differ (20). Thus, mea-
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surements of O4-etT levels in target or surrogate cells (e.g., obtained by sputum induction) may identify those individuals among smokers which are highly exposed or are unable to repair this type of DNA damage. As those subjects are likely to be at an increased risk for developing smoking-related malignancies, once identified, they could be subjected to preventive measures.
Acknowledgment. Dr. Roger Godschalk was a recipient of a visiting scientist fellowship awarded by the DKFZ in 1999. The authors would like to thank Dr. H. Besarati-Nia and Dr. F. J. Van Schooten (Maastricht University, The Netherlands) for providing induced sputum samples and Dr. W. Hull (German Cancer Research Center, Heidelberg, Germany) for NMR analyses. Dr. R. Owen (German Cancer Research Center, Heidelberg, Germany) is acknowledged for helpful scientific discussions.
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