Detection and Quantitation of Acrolein-Derived 1,N²-Propanodeoxyguanosine Adducts in Human Lung by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

Abstract

Acrolein, a widely distributed environmental pollutant, reacts with dGuo in DNA to form two pairs of 1,N²-propano-dGuo adducts: (6R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (α-OH-Acr-dGuo) and (8R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (γ-OH-Acr-dGuo). α-OH-Acr-dGuo is the more mutagenic and induces mainly G→T transversions. A recent study demonstrated that acrolein DNA adducts are preferentially formed in p53 mutational hotspots in human lung cancer, but there are no reports on the presence of these adducts in human lung. To directly investigate this question, we have developed a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for quantitative analysis of Acr-dGuo adducts in DNA. Our method is based on the enzymatic hydrolysis of DNA isolated from human lung in the presence of [¹³C₁₀,¹⁵N₅]Acr-dGuo as internal standards. Acr-dGuo adducts are enriched from the hydrolysates by solid phase extraction and analyzed by LC-ESI-MS/MS using selected reaction monitoring. The method is accurate and precise, and the identity of the adducts was confirmed by monitoring different transitions from the same parent ion, and by carrying out reactions with NaOH and NaBH₄, which produced N²-(3-hydroxypropyl)dGuo or 1,N²-(1,3-propano)dGuo from γ-OH-Acr-dGuo and α-OH-Acr-dGuo, respectively. Thirty DNA samples from lung tissue were analyzed and Acr-dGuo adducts were detected in all samples. Both α-OH- and γ-OH-Acr-dGuo were observed in most of the samples; total adduct concentrations ranged from 16 – 209 adducts/10⁹ nucleotides. These results demonstrate for the first time that both types of Acr-dGuo adducts are present in human lung DNA. There was no difference in adduct levels between current and ex-smokers. Collectively, the results support a plausible role for acrolein as one cause of p53 mutations in human lung.

Introduction

Acrolein, a highly reactive α,β-unsaturated aldehyde, is a widely distributed environmental pollutant (1) and is also formed endogenously through lipid peroxidation (2). Its concentration in cigarette smoke is relatively high, about 18 - 98 μg per cigarette (3). It is mutagenic in bacteria (4–6) and in cultured human cells (7,8). However, it is generally considered non-carcinogenic, except that one study reported the induction of bladder tumors in rats treated with acrolein (9,10). The weak carcinogenicity of acrolein may be due to efficient detoxification by glutathione or other sulfhydryls. Nonetheless, acrolein is strongly suspected to be responsible for the induction of secondary bladder tumors in cyclophosphamide-treated patients (11).

Acrolein reacts readily with dGuo in DNA to form cyclic 1,N²-propanodeoxyguanosine adducts (Acr-dGuo, Scheme 1). Depending on the direction of the initial Michael addition, two pairs of stereoisomers of (6R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one (α-OH-Acr-dGuo) and (8R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)one (γ-OH-Acr-dGuo) are formed (12). α-OH-Acr-dGuo in particular is mutagenic in human cells and induces predominantly G→T transversions (8). Acr-dGuo adducts have been detected in various human tissues as well as tissues from untreated animals (13–16). Early studies used ³²P-postlabeling coupled with HPLC and detected γ-OH-Acr-dGuo as the major adduct in vivo. Its level was 3-fold higher in oral tissues of cigarette smokers compared with non-smokers (15). Although extremely sensitive, ³²P-postlabeling lacks internal standards for quantitation. A capillary liquid chromatography nanoelectrospray isotope dilution tandem MS method was recently developed for analysis of γ-OH-Acr-dGuo in DNA hydrolysates (16). Its levels were significantly higher in brain tissues from Alzheimer’s disease subjects compared with age-matched controls.

Scheme 1.

Structures of Acr-dGuo adducts.

A recent study demonstrated that acrolein DNA adducts are preferentially formed at p53 mutational hotspots in human lung cancer and inhibit DNA repair (17). This study challenged the hypothesis that the p53 mutations are due to reactions with polycyclic aromatic hydrocarbon diol epoxides. These results raise the possibility that acrolein, which occurs in quantities up to 10,000 times as great as benzo[a]pyrene in cigarette smoke, may be a major etiological agent for cigarette smoking-related lung cancer. However, there are no reports in the literature on the presence of acrolein-DNA adducts in human lung tissue. In a previous study, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to analyze the crotonaldehyde- and acetaldehyde-derived 1,N²-propanodeoxyguanosine (Cro-dGuo), a structurally related adduct (18).

In the present study, we investigated Acr-dGuo adducts in human lung DNA using a similar method. The results demonstrate their presence in relatively high quantities.

Experimental Section

HPLC-UV analysis

This was carried out using Waters Associates (Milford, MA) instruments equipped with a UV detector (Shimadzu Scientific Instruments, Columbia, MD) operated at 254 nm or a model 996 photodiode array detector. System 1 used a 4.6 mm × 25 cm 5 μm Luna C18 column (Phenomenex, Torrance, CA) with isocratic elution by 5% acetonitrile in H₂O at a flow rate of 0.7 mL/min. This system was used for the purification of Acr-dGuo and [¹³C₁₀,¹⁵N₅]Acr-dGuo. System 2 used the same column as system 1 with a gradient from 5 to 40% CH₃OH in H₂O over the course of 35 min at a flow rate of 0.7 mL/min. This system was used for the analysis of dGuo.

Chemicals and Enzymes

[¹³C₁₀,¹⁵N₅]dGuo was obtained from Spectra Stable Isotopes (Columbia, MD). Ethanol was obtained from AAPER Alcohol and Chemical Co. (Shelbyville, KY). 2-Propanol was purchased from Acros Organics (Morris Plains, NJ). Puregene DNA purification solutions were procured from Gentra Systems (Minneapolis, MN). Calf thymus DNA, micrococcal nuclease, and phosphodiesterase II were obtained from Worthington Biochemical Co. (Lakewood, NJ). Alkaline phosphatase was obtained from Roche Diagnostics Corporation (Indianapolis, IN). All other chemicals were purchased from Sigma-Aldrich.

Acr-dGuo and [¹³C₁₀,15N₅]Acr-dGuo

Acr-dGuo standards were prepared as described (12) from the reaction of dGuo and acrolein. In brief, acrolein (56 mg, 1 mmol) was allowed to react with dGuo (25 mg, 0.09 mmol) in 10 mL of 0.1 M phosphate buffer (pH 7) at 37 ºC overnight. The products were purified by HPLC system 1 (15). α-OH-Acr-dGuo eluted earlier as two interchanging peaks of equal height, corresponding to the two diastereomers, while γ-OH-Acr-dGuo eluted as a single peak. The two peaks of α-OH-Acr-dGuo were collected together, and both α-OH- and γ-OH-Acr-dGuo were characterized and quantified by ¹H NMR, using toluene as an internal standard. The NMR spectra were consistent with published results (11, see Supporting Information). α-OH-Acr-dGuo: UV λ_max (ε) 259 nm (16800); positive ESI-MS m/z 324 [M + H]⁺; MS/MS of m/z 324 (collision energy 30 eV): m/z (relative intensity) 208 [BH]⁺ (100), 190 [BH - H₂O]⁺ (53), 152 [Gua + H]⁺. γ-OH-Acr-dGuo: UV λ_max (ε) 259 nm (18000); positive ESI-MS m/z 324[M + H]⁺; MS/MS of m/z 324 (collision energy 30 eV): m/z (relative intensity) 208 [BH]⁺ (48), 190 [BH - H₂O]⁺ (29), 164 [BH - CH₃CHO]⁺ (100), 152[Gua + H]⁺(18), 135 [Gua- NH₃ + H]⁺ (13). The approximate yields were 5% for each isomer. [¹³C₁₀,¹⁵N₅]Acr-dGuo was prepared the same way from [¹³C₁₀,¹⁵N₅]dGuo and quantified by UV at 254 nm. The amount of Acr-dGuo in [¹³C₁₀,¹⁵N₅]Acr-dGuo, as determined by LC-MS/MS, was less than 0.5%.

Human tissue samples

This study was approved by the University of Minnesota Research Subjects’ Protection Programs Institutional Review Board Human Subjects Committee. Thirty lung samples were obtained from The Cancer Center Tissue Procurement Facility. The samples were histologically confirmed as normal tissue. They were obtained at surgery, immediately frozen in liquid N₂, and stored at -80 ºC until DNA isolation. Urine samples were also obtained from some subjects just prior to surgery. They were analyzed for nicotine and cotinine as described previously (19).

DNA Isolation

This was performed as previously described (20), following the “DNA Purification from 1 g Animal Tissue” protocol (Gentra Systems) with several modifications. Isolated DNA was stored at −20 ºC until sample preparation. For the artifact study, lung tissue samples were split into 2 portions. One was isolated as usual, and the other was homogenized in cell lysis solution containing 100 mM NaBH₃CN. For this portion of the sample, isopropanol, ethanol, and 70% ethanol all contained 100 mM NaBH₃CN.

Analysis of DNA for Acr-dGuo

For enzymatic hydrolysis, DNA (0.1 – 1.0 mg) was dissolved in 900 μL of 10 mM sodium succinate/5 mM CaCl₂ buffer (pH 7.0) to which 25 fmol of each isomer of [¹³C₁₀,¹⁵N₅]Acr-dGuo was added as internal standard. The mixture was heated at 100 ºC for 30 min and cooled to room temperature. Enzymatic hydrolysis was performed by incubation with 75 units of micrococcal nuclease (from Staphylococcus aureus) and 0.45 unit of phosphodiesterase II (from bovine spleen) at 37 ºC for 6 h. Then, 150 units of alkaline phosphatase (from calf intestine) were added, and the mixture was incubated at 37 ºC overnight. A 10 μL aliquot was removed for dGuo quantitation, and the remaining hydrolysate was purified using a solid phase extraction (SPE) cartridge [Strata-X, 33 μm, 30 mg/1 mL (Phenomenex)]. After the sample was applied, the cartridge was washed with 1 mL H₂O and 1 mL 5% CH₃OH/H₂O, and the analyte was eluted with 1 mL 70% CH₃OH/H₂O. The eluants were evaporated to dryness, and dissolved in 20 μL of H₂O for LC-ESI-MS/MS analysis. A buffer control which lacked DNA was prepared each time and processed in the same way to exclude any contamination; while a calf thymus DNA (0.5 mg) sample was prepared and served as a positive control.

LC-ESI-MS/MS analysis was carried out with an Agilent 1100 capillary flow HPLC (Agilent Technologies, Palo Alto, CA) equipped with a 100 mm × 0.5 mm 1.8 μm particle size C18 column (Agilent Zorbax SB-C18) and coupled to either a Finnigan Quantum Ultra AM or Discovery Max (ThermoElectron, San Jose, CA) triple quadrupole mass spectrometer. The solvent elution program was a gradient from 5 to 25% CH₃OH in 15 mM ammonium acetate buffer in 20 min at a flow rate of 10 μL/min at 50 ºC. The ESI source was set in the positive ion mode as follows: voltage, 3.7 kV; current, 3 μA; and heated ion transfer tube, 275 ºC. The adducts were analyzed by MS/MS using selected reaction monitoring (SRM). Ion transitions of m/z 324 → m/z 208 (Acr-dGuo) and m/z 339 → m/z 218 ([¹³C₁₀,¹⁵N₅]Acr-dGuo) with collision energy of 12 eV were used for quantitation and those of m/z 324 → m/z 164 and m/z 324 → m/z 190 (Acr-dGuo) and m/z 339 → m/z 174 and m/z 339 → m/z 200 ([¹³C₁₀,¹⁵N₅]Acr-dGuo) with collision energy of 32 eV were used for structural confirmation. Other MS parameters were optimized to achieve maximum signal intensity.

Calibration curves were constructed before each analysis using standard solutions of Acr-dGuo and [¹³C₁₀,¹⁵N₅]Acr-dGuo. A constant amount of [¹³C₁₀,¹⁵N₅]Acr-dGuo (10 fmol) was mixed with differing amounts of Acr-dGuo (0.5 – 100 fmol) and analyzed by LC-ESI-MS/MS-SRM. dGuo content was determined by HPLC system 2, and total nucleotides calculated from the amount of dGuo, considering that dGuo accounts for 19.9% of the nucleotides in human DNA (21). The adduct levels were expressed per 10⁹ nucleotides.

Reaction of Acr-dGuo with NaOH and NaBH₄

The eluant from SPE containing the adducts was dissolved in 0.5 mL of 0.5 N NaOH containing an excess of NaBH₄. The resulting mixture was heated at 100 °C for 1 h, cooled to room temperature, and neutralized to pH 7 with 1 N HCl. The mixture was loaded on another Strata-X SPE cartridge and washed with 2 mL of H₂O to remove salts. The corresponding products were eluted by 1 mL 70% CH₃OH/H₂O and analyzed by LC-ESI-MS/MS, with the following ion transitions: m/z 326 → m/z 210 [N²-(3-hydroxypropyl)-dGuo], m/z 341 → m/z 220 {[¹³C₁₀,¹⁵N₅]N²-(3-hydroxypropyl)-dGuo}, m/z 308 → m/z 192 [1,N²-(1,3-propano)-dGuo], and m/z 323 → m/z 202 {[¹³C₁₀,¹⁵N₅]1,N²-(1,3-propano)-dGuo}.

Results

Standard characterization and calibration curves

Acr-dGuo standards were prepared as described (12), and characterized by NMR (see Supporting Information). They exist as two regioisomers, α-OH- and γ-OH-Acr-dGuo. The two diastereomers of α-OH-Acr-dGuo eluted earlier on HPLC as two interchanging peaks, while γ-OH-Acr-dGuo eluted as a single peak. The internal standard for our analysis was [¹³C₁₀,¹⁵N₅]Acr-dGuo, prepared by reacting acrolein with [¹³C₁₀,¹⁵N₅]dGuo, and characterized by UV and LC-ESI-MS, and comparison to Acr-dGuo. For LC-ESI-MS/MS-SRM analysis, the transitions monitored were m/z 324 → m/z 208 for Acr-dGuo and m/z 339 → m/z 218 for [¹³C₁₀,¹⁵N₅]Acr-dGuo. Calibration curves were plotted for the concentration ratios versus the integrated peak area ratios of analyte and internal standards. The two peaks corresponding to α-OH-Acr-dGuo were both integrated to reflect the total amount of this isomer, and linear responses were observed for both α-OH- and γ-OH-Acr-dGuo.

Method Development and Validation

The method was developed based on our previous study on the analysis of Cro-dGuo adducts (18), with some modifications. DNA was enzymatically hydrolyzed in the presence of [¹³C₁₀,¹⁵N₅]Acr-dGuo as internal standard, and the analytes were enriched from the hydrolysate by SPE. The enzymatic hydrolysis procedures were optimized to reduce background noise and signal suppression in the MS analysis. Our previous method used DNase I, phophodiesterase I, and alkaline phosphatase with incubation at 37 °C for 1 h. When calf thymus DNA was hydrolyzed in this way, significant signal suppression was observed for Acr-dGuo, which affected quantitation. This was probably due to the polarity of Acr-dGuo, which eluted close to unmodified dAdo. Our current method employed heating DNA to 100 °C for 30 min, followed by the addition of micrococcal nuclease, phosphodiesterase II, and alkaline phosphatase, and overnight incubation (22). Heat-denatured DNA was hydrolyzed much more rapidly by micrococcal nuclease (23,24). In addition, alkaline phosphatase contained a small amount of adenosine deaminase, which converted dAdo to dIno during the longer incubation. When heating was omitted, the measured level of Acr-dGuo was much lower than when heating was included (data not shown). Overall, this method gave more complete hydrolysis of DNA and removed dAdo which interfered with the Acr-dGuo analysis. This resulted in lower background and less signal suppression. Also, various HPLC conditions were investigated for better separation of the three isomeric peaks of Acr-dGuo. LC-ESI-MS/MS-SRM chromatograms obtained upon analysis of untreated calf thymus DNA are shown in Figure 1 (Panel A). All three product peaks were observed in the transition m/z 324 → m/z 208 and they coeluted with the internal standards peaks in the transition m/z 339 → m/z 218. No peaks were observed at this retention time in a buffer control which lacked DNA (data not shown). When the collision energy was increased to 32 eV, a peak at the retention time of γ-OH-Acr-dGuo was observed in the transition m/z 324 → m/z 164 [BH - CH₃CHO]⁺, while all three peaks were observed in the transition of m/z 324 → m/z 190 [BH - H₂O]⁺. Identical peaks were observed for the internal standards at the corresponding transitions. These results were fully consistent with the MS/MS analysis of standards. To further investigate peak identity, eluants from SPE were treated with NaOH and NaBH₄. Under these conditions, α-OH- and γ-OH-Acr-dGuo react differently (Scheme 2). γ-OH-Acr-dGuo undergoes base-catalyzed ring-opening followed by reduction of the intermediate aldehyde, producing N²-(3-hydroxypropyl)-dGuo, while treatment of α-OH-Acr-dGuo with NaOH and NaBH₄ results in the elimination of H₂O followed by reduction, giving unsubstituted 1,N²-(1,3-propano)-dGuo (12). The results of analysis of calf thymus DNA after the reaction with NaOH and NaBH₄ are shown in Figure 1 (Panel B). The peaks in the transition m/z 324 → m/z 208 disappeared. By comparing with standards that underwent the same procedures, the peak eluting at 14.1 min in the transition m/z 308 → m/z 192 was assigned as 1,N²-(1,3-propano)-dGuo, which came from reduction of α-OH-Acr-dGuo, while the peak eluting at 12.3 min in the transition m/z 326 → m/z 210 was N²-(3-hydroxypropyl)-dGuo, which came from reduction of γ-OH-Acr-dGuo. Both peaks coeluted with peaks in the transitions m/z 323 → m/z 202 and m/z 341 → m/z 220, from the internal standards. These results unambiguously demonstrate the identity of the adducts as those shown in Scheme 1.

Figure 1.

Chromatograms obtained upon LC-ESI-MS/MS analysis of calf thymus DNA. Calf thymus DNA was enzymatically hydrolyzed, purified by SPE, and analyzed (panel A); or the eluants from SPE were treated with NaOH and NaBH₄ and analyzed (panel B).

Scheme 2.

Reaction of α-OH- and γ-OH-Acr-dGuo with NaOH and NaBH₄.

Accuracy was determined by analyzing calf thymus DNA spiked with differing levels of Acr-dGuo standards. Each sample was analyzed in triplicate. The results are summarized in Figure 2, which shows good agreement between expected and measured values. Precision of the method was investigated by analyzing calf thymus DNA in six replicates on three separate days. The interday CVs were 5% for α-OH-Acr-dGuo and 7% for γ-OH-Acr-dGuo, as summarized in Table 1. The limit of quantitation (LOQ) for pure standard was 0.5 fmol injected on column (S/N = 10) as well as a linear MS response. LOQ in DNA samples was estimated as 4 adducts/10⁹ nucleotides, starting from 0.5 mg of DNA. The recovery of 25 fmol of internal standard during sample processing was 88% and 84% for α-OH-Acr-dGuo and →-OH-Acr-dGuo, respectively. The stability of the analytes was investigated by analyzing 25 fmol of standards that were processed through the same enzymatic hydrolysis procedure, and no decomposition was observed.

Figure 2.

Relationship of added to detected Acr-dGuo. Various amounts of α-OH- and γ-OH-Acr-dGuo standards (17, 34, 85, 170, and 340 fmol) were added to calf thymus DNA (0.5 mg) and analyzed by the method described in the text. Background levels in calf thymus DNA (40 adducts/10⁹ nucleotides for α-OH-Acr-dGuo, and 30 adducts/10⁹ nucleotides for γ-OH-Acr-dGuo) were subtracted from each amount detected. Each point represents a triplicate measurement. A, α-OH-Acr-dGuo, R² = 1.0; B, γ-OH-Acr-dGuo, R² = 1.0.

Table 1.

Precision of the LC-ESI-MS/MS method for analysis of Acr-dGuo^a.

	Acr-dGuo level (adducts/109 nucleotides)
	Day 1 Mean ± SD	Day 2 Mean ± SD	Day 3 Mean ± SD	Average	%RSD
α-OH	40 ± 7	37 ± 6	43 ± 5	40 ± 3	7
γ-OH	30 ± 7	27 ± 3	29 ± 6	29 ± 1	5

^aSix aliquots of calf thymus DNA were analyzed on 3 separate days.

It was possible that Acr-dGuo adducts could have been formed as artifacts from the reaction of intracellular acrolein with DNA during DNA isolation. In this case, acrolein could be pre-existing in the cell or produced from lipid peroxidation during DNA isolation. We tested this possibility by adding NaBH₃CN to the DNA isolation solutions. NaBH₃CN would reduce any acrolein released during DNA isolation. Three lung tissue samples were each divided into two portions and DNA was isolated using solutions containing or not containing NaBH₃CN. In other respects, the enzymatic hydrolysis, SPE, and LC-MS/MS analysis procedures were identical. The results indicated that levels of both α-OH- and γ-OH-Acr-dGuo were similar under these two conditions, which eliminated the possibility of artifactual formation of Acr-dGuo adducts.

Analysis of Human Lung DNA for Acr-dGuo

Lung tissue samples were obtained at surgery from 30 subjects (Table 2). Ten were male, 19 were female, and gender information was not available for one subject. Ages ranged from 37 – 81 years (mean ± S.D. 62.2 ± 12), and the age of one subject was unknown. All subjects were current or ex-smokers, based on self report. Urinary nicotine and cotinine levels were available for 14 subjects. These analyses established that 5 of the subjects – numbers 1, 2, 8, 9, 10 – were current smokers, and disagreed with self-report for 2 subjects – numbers 8 and 13 (although we cannot exclude the possibility that the subjects with positive urinary cotinine and nicotine may have been using nicotine replacement therapy). Acr-dGuo adducts were found in all samples. Figure 3 shows a representative chromatogram. Five DNA samples were also analyzed after reaction with NaOH and NaBH₄, which gave similar chromatograms to those shown in Figure 1, Panel B. Both α-OH- and γ-OH-Acr-dGuo were detected in all samples except one in which only γ-OH-Acr-dGuo was observed. Adduct levels ranged from ND – 154 adducts/10⁹ nucleotides for α-OH-Acr-dGuo and 6.4 – 159 adducts/10⁹ nucleotides for γ-OH-cr-dGuo, which were much higher than the Cro-dGuo levels we analyzed before (18). There was no difference in adduct levels between confirmed current smokers (N = 5) and non-smokers (N = 9), nor was there any relationship of adduct levels to self-reported time since cessation of smoking, gender, or age.

Table 2.

Levels of Acr-dGuo detected in human lung DNA.

					urinary biomarkers			Acr-dGuo levelc (adducts/109 nucleotides)
subject	gender	race	age at surgery	self-reported smoking historya	cotinine (ng/ml)	nicotine (ng/ml)	amount of DNA analyzed (mg)b	α-OH	γ-OH	total
1	F	W	60	current	652	115	0.14	91	47	138
2	F	W	47	current	5084	1462	0.51	45	21	66
3	F	W	52	2 yr	6.8	1.3	1.25	18	9	27
4	M	W	69	2 yr	25.2	4.5	1.09	18	10	27
5	F	W	72	10 yr	3.7	0.7	0.20	25	49	74
6	F	W	79	10 yr	9.2	1.3	0.10	78	49	127
7	M	W	66	26 yr	4.3	1.5	0.03	154	55	209
8	F	W	51	3 months	2397	534	0.70	10	6	16
9	M	W	53	current	2346	410	0.81	15	10	25
10	M	W	53	current	692	117	0.54	ND	159	159
11	F	W	61	9 yr	26.7	4.1	0.29	75	47	121
12	F	B	52	6 yr	34.1	16.4	0.55	25	12	36
13	F	W	37	current	3.3	1.7	0.61	16	9	25
14	M	W	78	24 yr	7.2	0.6	0.09	85	53	138
15	F	W	68	1 month	NA	NA	0.65	33	23	56
16	F	W	72	2 months	NA	NA	0.49	20	13	33
17	M	W	78	NA	NA	NA	0.77	12	18	29
18	F	W	60	2 yr	NA	NA	0.10	44	24	68
19	M	W	72	7 yr	NA	NA	0.86	10	6	16
20	F	W	46	1 week	NA	NA	0.72	10	9	19
21	F	W	81	23 yr	NA	NA	0.48	17	17	34
22	F	W	58	18 yr	NA	NA	1.08	13	13	26
23	F	W	76	10 yr	NA	NA	0.04	110	82	192
24	M	W	66	17 yr	NA	NA	0.64	16	14	29
25	F	W	51	1 yr	NA	NA	0.53	10	13	24
26	F	W	56	1 yr	NA	NA	0.33	35	21	56
27	M	W	74	16 yr	NA	NA	0.79	15	11	26
28	M	W	69	9 yr	NA	NA	0.84	20	22	42
29	F	W	49	2 months	NA	NA	0.98	17	12	29
30	NAe	NA	NA	NA	NA	NA	0.04	111	47	158
Mean ± S.D.			62.2 ± 12				0.54 ± 0.35	40 ± 38	29 ± 31	68 ± 58

^aReported time since quitting

^bCalculated based on dGuo

^cEach value represents a single measurement. dGuo was determined by HPLC-UV.

^dND, not detected

^eNA not available

Figure 3.

Chromatograms obtained upon LC-ESI-MS/MS-SRM analysis of DNA from human lung.

Discussion

The results of this study demonstrate for the first time that acrolein-DNA adducts, including the mutagenic adduct α-OH-Acr-dGuo, are present in human lung. This is significant in view of a recent study which showed that acrolein produces a spectrum of DNA damage in the p53 gene that is remarkably similar to the spectrum of mutations found in this gene in lung tumors from smokers (17). The total levels of acrolein-DNA adducts quantified here, about 1 per 10⁷ nucleotides, are higher than “PAH-DNA adducts” reported in lung tissue by ELISA, typically about 0.3 adducts per 10⁷ nucleotides, and benzo[a]pyrene diol epoxide – DNA adducts, which are frequently undetectable and typically amount to 0.1 – 0.5 per 10⁷ nucleotides when they are detected (25–27). Collectively, these results indicate that acrolein could contribute significantly to the mutations seen in the p53 gene in lung cancer and challenge the widely held view that these mutations are due to polycyclic aromatic hydrocarbon diol epoxides (28). The weak or non-existent carcinogenicity of acrolein and the similarity in adduct levels between a limited number of confirmed current and ex-smokers on the other hand argue against a significant direct role for this compound in tobacco smoke - induced lung cancer, and further work is clearly needed to resolve these issues (29).

The structure of acrolein DNA adducts was first characterized in 1984 (12). Using ³²P-postlabeling/HPLC, Chung and co-workers have detected Acr-dGuo adducts in various human and untreated animal tissues (13–15), suggesting the existence of an endogenous source. γ-OH-Acr-dGuo was the major adduct detected, and the levels of α-OH-Acr-dGuo were too low to be quantified or detected in most tissues. ³²P-Postlabeling has certain limitations, such as the inability to provide unambiguous structural identification and the lack of internal standards for reliable quantitation. In the present study, we have developed a sensitive and specific LC-ESI-MS/MS method for the quantitative analysis of Acr-dGuo adducts. Compared with the method described by Liu et al (16), ours does not involve the setup of a nanospray source for the mass spectrometer, and uses commercially available capillary columns. Although the sensitivity is slightly lower than the previously published method, it is still sufficient to detect these adducts in human tissues.

The levels of Acr-dGuo in human lung were higher than the structurally related Cro-dGuo adducts which we have analyzed before (18). This is consistent with previous studies by Chung and co-workers, who also detected higher levels of Acr-dGuo than Cro-dGuo (13,14). Cigarette smoking is not the only source of these adducts. Acrolein from the environment and from endogenous formation through lipid peroxidation should also contribute. As reported by Pan and Chung (30), the rate of Acr-dGuo formation from polyunsaturated fatty acids under oxidative conditions was much higher than that of Cro-dGuo, consistent with our results.

We failed to observe a relationship between levels of Acr-dGuo adducts and urinary nicotine and cotinine, or time since cessation of smoking based on self-report. However, nicotine and cotinine data were available only for a small number of subjects, and self-report can be unreliable. Nothing is known about the kinetics of Acr-dGuo adduct removal after smoking cessation. Exposures to acrolein other than cigarette smoking as well as endogenous formation of acrolein from lipid peroxidation could contribute to adduct levels and will vary among people. Furthermore, individuals will have different abilities to detoxify acrolein and to repair Acr-dGuo adducts. Further research is needed to establish the relationship, if any, of Acr-dGuo adducts to cigarette smoking.

In summary, we have developed a sensitive and specific MS method for the quantitative analysis of Acr-dGuo adducts in human tissue DNA. Our results clearly demonstrate the presence of Acr-dGuo adducts in human lung DNA. Additional research is required to assess the contribution of acrolein to lung cancer caused by cigarette smoking.