University of Minnesota Cancer Center, Minneapolis, MN 55455, USA
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Abstract |
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Abbreviations: GC-NICI-MS/MS, gas chromatography-negative ion chemical ionization-tandem mass spectrometry; GC-NICI-MS-SIM, gas chromatography-negative ion chemical ionization-mass spectrometry with selected ion monitoring; N-ethylvaline-PFPTH, 1-ethyl-5-isopropyl-3-pentafluorophenyl-2-thiohydantoin; N-methylvaline-PFPTH, 1-methyl-5-isopropyl-3-pentafluorophenyl-2-thiohydantoin; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; PAH, polycyclic aromatic hydrocarbons.
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Introduction |
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Cigarette smoke carcinogens are the likely cause of cancer in smokers (6). Some such as N-nitrosamines and polycyclic aromatic hydrocarbons (PAH) require metabolic activation while others such as acetaldehyde and ethylene oxide do not. In either case, smokers are exposed to a constant flux of electrophiles which react with DNA to produce adducts. Considerable data indicate that these adducts cause permanent mutations in critical genes such as ras and p53, leading to loss of normal cellular growth control and ultimately to cancer (7,8). DNA adducts of tobacco-specific nitrosamines, PAH, aromatic amines, ethylene oxide and of unknown origin have been detected in tissues of smokers. However, technical problems have hindered these studies and relatively few DNA adducts have been convincingly characterized and quantified in smokers (G.P.Pfeifer et al., submitted for publication).
Electrophiles, which react with DNA, also react with proteins such as hemoglobin and albumin. Hemoglobin adducts are potential surrogates for quantifying DNA damage by carcinogens (9,10). Advantages of hemoglobin adducts as dosimeters of carcinogen damage include the ease with which large quantities of hemoglobin are obtained, the relatively long lifetime of the red cell which permits accumulation of adducts, and the lack of any known repair mechanisms. Hemoglobin adducts of tobacco-specific nitrosamines, PAH, aromatic amines, ethylene oxide and other carcinogens have been quantified in smokers (1017).
Two relatively recent studies demonstrated higher levels of 3-ethyladenine in the urine of smokers than in non-smokers (18,19). This could result from ethylation of adenine in DNA followed by depurination and excretion of 3-ethyladenine. The results of these studies suggest the presence in cigarette smoke of an unknown DNA ethylating agent, but there are no other data in the literature that support this potentially important proposal. Ethyl adducts in DNA, such as O6-ethyldeoxyguanosine and O4-ethylthymidine, have miscoding properties and are closely associated with cancer in animal studies (2022). If smoking results in increased DNA ethylation, then increased hemoglobin ethylation also should be expected. Therefore, in this study, we analyzed hemoglobin of smokers and non-smokers for ethylated N-terminal valine. Methods for determining alkylated N-terminal valine of hemoglobin have been developed and applied by Tornqvist et al. (16,23,24), but there is only one report on ethylation. In that study, levels were below the detection limit of the method (24). We modified Tornqvist's method to increase its selectivity and sensitivity for detection of N-terminal N-ethylvaline. We also analyzed for N-terminal N-methylvaline in hemoglobin of smokers and non-smokers.
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Materials and methods |
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Non-smokers were participants in a study that assessed the effects of diet on N-terminal N-alkylvalines in hemoglobin (25). On two occasions, 1 month apart, each subject provided a 10 ml blood sample, collected in a tube containing EDTA. Samples from the first blood draw of 29 non-smokers were randomly chosen for the analyses. An additional nine samples were randomly chosen from the second blood sampling of these 29 subjects. The total number of non-smokers in the diet study was 114, and analysis of N-terminal N-ethylvaline and N-methylvaline in the hemoglobin of these subjects has been described (25). The method used in that study for N-terminal N-ethylvaline was different from the one described here, as it employed GC-NICI-MS-SIM, not GC-NICI-MS/MS. When the current improved method for N-terminal N-ethylvaline became available, all samples in the earlier study were re-analyzed by GC-NICI-MS/MS, without any effect on the conclusions. A subset of the 114 non-smoker samples, the ones described here, were analyzed for N-terminal N-ethylvaline at the same time and by the same method as the smokers' samples described here. Therefore, only this subset was used in the present comparison. (The worked up fractions of the remaining non-smoker samples had been frozen for an additional 6 months prior to GC-NICI-MS/MS analysis for N-ethylvaline-PFPTH and the results were therefore not included in the present comparison with smokers. However, inclusion of those results did not change the conclusions of the present study.) All N-terminal N-methylvaline analyses were by the same method, GC-NICI-MS-SIM. In this paper, we only present data for the same non-smokers for whom we report measurements of N-terminal N-ethylvaline. Inclusion of all data did not change the conclusions reported here.
Both studies were approved by the University of Minnesota Research Subjects' Protection Programs Institutional Review Board Human Subjects Committee.
Chemicals
Formamide (USB, Cleveland, OH), hexane (Fisher Scientific, Pittsburgh, PA), dodecane (Fisher Scientific), pentafluoro-phenyl isothiocyanate (Fluka, Ronkonkoma, NY) [D3]N-methyl-valine-HLTPF (SynPep, Dublin, CA), and N-methylvaline (Sigma Chemical Co., St Louis, MO) were purchased. N-Ethylvaline, 1-ethyl-5-isopropyl-3-pentafluorophenyl-2-thio-hydantoin (N-ethylvaline-PFPTH), 1-methyl-5-isopropyl-3-pentafluorophenyl-2-thiohydantoin (N-methylvaline-PFPTH), [D3]N-methylvaline, [D3]N-methylvaline-PFPTH, and 1-methyl-5-isopropyl-3-phenyl-2-thiohydantoin were synthesized essentially as described by Rydberg et al. (26).
Analysis of N-terminal N-ethylvaline and N-methylvaline in globin
Red cells were separated from serum immediately after collection by centrifugation at 1000 g for 15 min at 4°C. The red cells were washed twice with 20 ml saline (0.9% in H2O) and stored at 20°C until analysis. For isolation of globin, 2 ml of red cells were lysed with 3 ml H2O in 30 ml Nalgene centrifuge tubes (Nalge Co., Rochester, NY) and 23 ml 0.5% HCl in cold isopropanol was added, and the mixture was agitated. The mixture was centrifuged at 3000 g for 20 min at 4°C. The acidified hemoglobin supernatant was transferred into a 50 ml centrifuge tube and globin was precipitated by slow addition, with mixing, of 20 ml cold ethyl acetate. The mixture was allowed to stand in ice for 20 min, then centrifuged to separate the precipitate. The precipitate was washed twice with ethyl acetate and once with pentane, then dried under a stream of N2. Globin was stored at 20°C until analysis. Just prior to analysis, the globin was dried in vacuo for 16 h.
Globin (20 mg) was dissolved in 1.5 ml formamide in a 5 ml glass centrifuge tube. The formamide was purified before use by extraction with two equal volumes of pentane (16). The globin samples were vortexed for 1 h for complete dissolution. The samples were then neutralized by addition of about 25 µl 1 N NaOH. The peptide [D3]N-methylvaline-HLTPF (5 ng, 6.85 pmol) was added to each sample as internal standard. The internal standard was initially dissolved in DMSO, then diluted in formamide. After addition of the internal standard, 7 µl of pentafluorophenyl isothiocyanate was added and the samples were shaken overnight at room temperature. The samples were then heated at 45°C for 90 min to complete the reaction. The pH was adjusted to 6.57.0 with 1015 µl 1 N NaOH. (During the derivatization reaction, the pH drops by ~1 U, which favors splitting of the derivative from the globin. Readjusting the pH to 7 minimizes the formation of emulsions during subsequent extractions.) The samples were extracted three times with 2.5 ml of ether. The extracts were combined into a 5 µl centrifuge tube and concentrated to dryness under a stream of N2. The residue was re-dissolved in 1 ml hexane and washed twice with 2.5 ml H2O, twice with 3 µl freshly prepared 0.1 M sodium carbonate, and again twice with H2O. The hexane layers were centrifuged for 10 min and all H2O was removed. Dodecane (5 µl) was added, the tubes were placed in a beaker of warm H2O, and the hexane was removed under a gentle stream of N2. The tubes were removed at the point of near dryness. The extracts were then transferred to 200 µl HPLC autosampler microvials with 3x65 µl 95% hexanes/5% isopropanol. The samples were stored at 20°C until HPLC purification.
A normal phase Pinnacle 5 mm cyano HPLC column (4.6x250 mm, Restek, Bellefonte, PA) with a cyano guard cartridge (3.0x4 mm i.d., Phenomenex, Torrance, CA) was used for further purification of the samples. Detection was by UV at 254 nm. Solvent A was 100% hexane and solvent B was 95% hexane:5% isopropanol. The flow rate was 1 ml/min.
1-Methyl-5-isopropyl-3-phenyl-2-thiohydantoin (0.16 mg) was injected to calibrate the system and determine the appropriate collection window. It eluted at 15.6 min. The solvent program was 100% A for 10 min, then from 100% A to 100% B in 20 min, then return to 100% A. Eluant was collected from 614.5 min, which included N-ethylvaline-PFPTH (retention time, 7.6 min), N-methylvaline-PFPTH (9.6 min), and [D3]N-methylvaline-PFPTH (9.8 min).
Dodecane (10 µl) was added to each 15 ml tube containing the collected HPLC fraction. The hexane was removed under a gentle stream of N2. The residue was transferred into a 200 µl microvial with 3x65 µl portions of hexane. The hexane was carefully evaporated under a gentle stream of N2. The samples, now in dodecane, were stored at 20°C until further analysis.
Gas chromatography-negative ion chemical ionization-tandem mass spectrometry (GC-NICI-MS/MS) was carried out on a Finnigan TSQ 7000 instrument (Thermoquest, San Jose, CA) interfaced with a CTC A200SE autosampler (Leap Technologies, Carrboro, NC) and a HP5890 series II GC (Agilent Technologies, Palo Alto, CA). The GC was fitted with a 30 mx0.32 mm x0.25 µm film thickness DB-1301 column (Agilent Technologies) and a 2 mx530 µm uncoated deactivated fused silica precolumn. The carrier gas was He, at 1.4 ml/min constant flow. Two µl of each sample were injected via splitless injection with a gooseneck splitless liner. The splitless vent flow was 120 ml/min. The purge open time was 0.75 min. The injector temperature was 225°C. The MS interface temperature was 250°C. The GC oven was programmed as follows: 100°C for 2 min, then 10°C/min until 140°C, then 2°C/min until 165°C, then hold for 15 min, then 15°C/min until 250°C, then hold for 5 min.
CI source parameters were as follows: filament emission current, 300 µA; electron energy, 200 eV; ion source temperature, 150°C. Methane was the reagent gas. For the GC-NICI-MS/MS determination of N-ethylvaline-PFPTH, m/z 351 was selected, and scanned at 0.5 amu resolution, scan time 1.0 s. The collision gas was Ar, collision energy 20 eV. The transition m/z 351322 [(M-1)-C2H5], which was the largest daughter ion, was monitored. For [D3]N-methylvaline-PFPTH, the transition m/z 340
206 was monitored.
For analysis of N-methylvaline-PFPTH by gas chromatography-negative ion chemical ionization-mass spectrometry with selected ion monitoring (GC-NICI-MS-SIM), the reagent gas was methane. CI source parameters were as follows: filament emission current, 700 µA; electron energy, 150 eV, ion source temperature, 150°C. For N-methylvaline-PFPTH, m/z 337 (M-1) was monitored and for [D3]N-methylvaline-PFPTH, m/z 340 was monitored. The peak width was 0.5 amu and the scan time was 0.3 s.
Retention times were as follows: N-ethylvaline-PFPTH, 19.68 min; N-methylvaline-PFPTH, 19.37 min, [D3]N-methylvaline-PFPTH, 19.35 min. Standard curves for N-ethylvaline-PFPTH and [D3]N-methylvaline-PFPTH were generated by GC-NICI-MS/MS. Levels of N-ethylvaline-PFPTH in each sample were corrected for loss of deuterated internal standard. Levels of N-methylvaline-PFPTH were similarly calculated using GC-NICI-MS-SIM.
Statistical analysis
For nine of the non-smokers, analyses were carried out on two blood draws 1 month apart. When two values were available, these were averaged and the resulting mean values included with the rest of the non-smokers. When one of the two samples was non-quantifiable, the remaining value was used. For samples in which N-ethylvaline-PFPTH was not detected, we used half the detection limit. Levels of N-terminal N-ethylvaline and N-methylvaline in smokers versus non-smokers were compared using the two-sided two-sample t-test and the two-sided Wilcoxon rank-sum test. Levels of N-terminal N-methylvaline in non-smokers on two occasions were compared using a paired t-test. Age distributions of smokers and non-smokers were compared using the two-sided two-sample t-test. Gender distribution of smokers and non-smokers was compared using the chi-squared test. Effects of age and gender on adduct level differences in smokers and non-smokers were compared by multiple regression. Correlations of N-terminal N-methylvaline and N-ethylvaline with other parameters were evaluated using Excel regression programs.
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Results |
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A CI-MS of N-ethylvaline-PFPTH is shown in Figure 3A. For the GC-NICI-MS/MS analysis, we selected m/z 351 (M-H). The daughter ion spectrum of m/z 351 is illustrated in Figure 3B
. We selected the transition m/z 351
322 (M-H-C2H5) for the MS/MS analysis. GC-NICI-MS/MS analysis provided considerably greater sensitivity and cleaner chromatograms than were achieved by GC-NICI-MS-SIM analysis of N-ethylvaline-PFPTH, m/z 351. The internal standard produced [D3]N-methylvaline-PFPTH, which was monitored at m/z 340
206. For analysis of N-methylvaline-PFPTH, which was present in greater quantities, GC-NICI-MS-SIM, m/z 337 (M-H), was satisfactory. The internal standard was monitored at m/z 340.
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In some samples, co-eluting peaks prevented quantification of N-ethylvaline-PFPTH. This occurred in four of 45 smoker samples and in six of 38 non-smoker samples (from 29 non-smokers), and these samples were excluded from the analysis. In the non-smoker samples, five of the six instances occurred among the nine individuals from whom we drew blood on two occasions, and consequently we used the remaining value. Thus, we had 39 smoker data points and 28 non-smoker data points for N-ethylvaline-PFPTH. There were no co-eluting peaks, which prevented quantification of N-methylvaline PFPTH.
Among the non-smokers, data were available for N-terminal N-methylvaline in hemoglobin of nine subjects obtained on two occasions 1 month apart. Levels were (mean ± SD) 808 ± 172 pmol/g globin on the first occasion and 893 ± 212 pmol/g globin on the second. Analysis of the data demonstrated that there was no significant difference in individual values on the two occasions (P = 0.27). For N-terminal N-ethylvaline, only four values were available on both occasions, as mentioned above. Among these four pairs of values, there were three below the detection limit (two from one individual). Therefore, intraindividual variation in the non-smoker N-terminal N-ethylvaline data could not be analyzed.
Smokers ranged in age from 29 to 66 (mean ± SD, 47.5 ± 8.4) and were 50% male. They smoked from 16 to 38 cigarettes per day (mean 24.5 ± 5.4). Non-smokers ranged in age from 20 to 57 (mean 29.4 ± 9.2) and were 34% male. Smokers were significantly older than non-smokers (P < 0.001). There was no significant difference in gender among the smokers and non-smokers. Levels of N-terminal N-ethylvaline in smokers and non-smokers are summarized in Figure 6. The mean level in smokers, 3.76 ± 2.77 pmol/g globin (n = 39) was significantly higher than in non-smokers, 2.50 ± 1.65 (n = 28), P = 0.023 (two-sided t-test) and P = 0.025 (Wilcoxon two-sided test). The difference was still significant after correction for gender and age (P = 0.019). Levels of N-terminal N-methylvaline in smokers and non-smokers are summarized in Figure 7
. The mean level in smokers, 997 ± 203 pmol/g globin (n = 45), was slightly higher than in non-smokers, 904 ± 149 pmol/g globin (n = 29). This difference was of borderline significance, P = 0.037 (two-sided t-test) and P = 0.071 (Wilcoxon two-sided test). When corrected for gender and age, the difference between smokers and non-smokers was not significant (P = 0.21).
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Discussion |
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There are several potential sources of an ethylating agent in cigarette smoke. One is nitrosation of ethylamine. Mainstream smoke contains relatively high levels of nitrogen oxides, 100600 µg/cigarette (29). The nitrosating potential of cigarette smoke has been demonstrated by studies, which found higher levels of nitrosamino acids in the urine of smokers compared with non-smokers (3032). Smoke also contains ~1 µg ethylamine/cigarette (33). Nitrosation of ethylamine would produce the ethyl diazonium ion, a well-known ethylating agent. Ethyl diazonium ion is also formed upon hydrolysis of N-nitroso-N-ethylurea or metabolic -hydroxylation of N-nitrosodiethylamine or N-nitrosoethylmethylamine (34). These possibilities seem less likely because N-nitroso-N-ethylurea is not known to be present in cigarette smoke and N-nitrosodiethylamine and N-nitrosoethylmethylamine occur in relatively low concentrations, 3 and 13 ng/cigarette, respectively (29). Nitrosation of ethanol could produce ethyl nitrite, another potential nitrosating agent. There are presently no reports of ethyl nitrite in cigarette smoke. Another possible source is acetaldehyde, which is found in smoke at levels of ~1 mg/cigarette, and is the primary metabolite of ethanol (35). Acetaldehyde could react with N-terminal valine to produce a Schiff base, which would then be required to undergo endogenous reduction to give N-terminal N-ethylvaline. Some support for this proposal can be found in the studies of Tornqvist et al. (36) who noted increased levels of N-terminal N-methylvaline in mice treated with formaldehyde. It is not clear how reduction would occur. It also appears unlikely that acetaldehyde could give rise to 3-ethyladenine, although there are reports that N2-ethyldeoxyguanosine is elevated in individuals who consumed ethanol (37). There was no correlation between alcohol consumption and N-terminal N-ethylvaline levels in the present study.
Levels of N-terminal N-methylvaline were ~10% higher in smokers than in non-smokers, a difference of borderline statistical significance, which disappeared when corrected for age and gender. Tornqvist et al. (36) found 540 pmol/g Hb N-methylvaline in smokers compared to 500 pmol/g Hb in non-smokers in a small study. A second investigation by this group examined N-terminal N-methylvaline levels in a group of monozygotic twins discordant for smoking (38). They found 24% higher levels in smokers, a significant difference. Bader et al. (39) found higher levels of N-terminal N-methylvaline in smokers than in non-smokers. Overall, there may be some increase in N-terminal N-methylvaline in smokers compared with non-smokers. There is clearly a high endogenous level of N-terminal N-methylvaline in globin of humans and other species. This can also be seen in the present results, where levels of N-terminal N-methylvaline were ~250350 times greater than those of N-terminal N-ethylvaline. Any effect of smoking would be seen only above the endogenous levels. Tornqvist et al. (36,38) argue that S-adenosyl methionine is the major source of endogenous N-terminal N-methylvaline. Other potential sources are methyl chloride, N-nitrosodimethylamine, and formaldehyde but the contribution of these agents appears to be less probable (36). Enzymatic methylation of proteins is well established but there are no reports on methylation of N-terminal valine of hemoglobin (40,41). Methylating nitrosamines such as the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosodimethylamine would be two likely sources of additional hemoglobin methylation in smokers (42). Measurements of 3-methyladenine in urine are complicated by its presence in diet and by background levels. Nevertheless, two controlled studies demonstrated increased excretion of 3-methyladenine in the urine of smokers (18,19). Mixed results have been obtained in studies of 7-methylguanine in DNA from smokers and non-smokers (43).
Representative data for some carcinogen hemoglobin adducts which have been quantified in smokers are summarized in Table I. In most cases, higher adduct levels are found in smokers than in non-smokers. Adduct levels range from 18 fmol/g globin (3-aminobiphenyl) to 997 000 fmol/g globin (N-methylvaline) in smokers. The carcinogens which produce relatively lower adduct levels such as NNK, N'-nitrosonornicotine, 3- and 4-aminobiphenyl, and benzo[a]pyrene are generally present in tens or hundreds of nanograms per cigarette, and all require metabolic activation prior to binding (44). Carcinogens which produce higher adduct levels such as acrylonitrile and ethylene oxide are present in microgram amounts per cigarette and do not require metabolic activation. The compounds producing lower hemoglobin adduct levels are generally stronger carcinogens in laboratory animals than those producing higher adduct levels, suggesting that reactions with protein may be protective, perhaps by competing with DNA reactions. Levels of N-ethylvaline are intermediate between these two groups. However, there are also substantial amounts of N-ethylvaline adducts in non-smokers indicating the presence of a relatively abundant non-tobacco related precursor.
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A limitation of this study was that the smokers and non-smokers were selected from different groups. The smoker samples were selected from a study investigating the effects of reduction in smoking on carcinogen uptake. The non-smoker samples were from a study investigating the effects of diet on hemoglobin adduct formation. A better design would have been to recruit matched groups of smokers and non-smokers. Nevertheless, N-terminal N-ethylvaline levels were still higher in smokers than in non-smokers, when corrected for differences in gender and age. Larger studies designed specifically to address this question will be required to confirm our results.
In summary, the results of this study demonstrate that ethylation of N-terminal valine of hemoglobin is ~50% higher in smokers than in non-smokers, a significant difference. These results are consistent with previous studies, which demonstrate increased excretion of 3-ethyladenine in the urine of smokers. The results indicate the presence in cigarette smoke of an as yet unidentified ethylating agent, which could contribute to the carcinogenic properties of smoke.
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Acknowledgments |
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