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. Author manuscript; available in PMC: 2020 Apr 15.
Published in final edited form as: Chem Res Toxicol. 2019 Feb 15;32(4):708–717. doi: 10.1021/acs.chemrestox.8b00374

Quantification of DNA Lesions Induced by 4‑(Methylnitrosamino)-1-(3-pyridyl)-1-butanol in Mammalian Cells

Su Guo †, Jiapeng Leng ‡, Ying Tan †, Nathan E Price ‡, Yinsheng Wang †,‡,*
PMCID: PMC6469357  NIHMSID: NIHMS1018807  PMID: 30714728

Abstract

Quantitative measurement of DNA adducts in carcinogen-exposed cells provides the information about the frequency of formation and the rate of removal of DNA lesions in vivo, which yields insights into the initial events of mutagenesis. Metabolic activation of tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its reduction product 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), leads to pyridyloxobutylation and pyridylhydroxybutylation of DNA. In this study, we employed a highly robust nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (nLC-nESI-MS/MS) coupled with the isotope-dilution method for simultaneous quantification of O6-[4-(3-pyridyl)-4-hydroxylbut-1-yl]-2′-deoxyguanosine (O6-PHBdG) and O2- and O4-[4-(3-pyridyl)-4-hydroxylbut-1-yl]-thymidine (O2-PHBdT and O4-PHBdT). Cultured mammalian cells were exposed to a model pyridylhydroxybutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (NNALOAc), followed by DNA extraction, enzymatic digestion, and sample enrichment prior to nLC-nESI-MS/MS quantification. Our results demonstrate, for the first time, that O4-PHBdT is quantifiable in cellular DNA and naked DNA upon NNALOAc exposure. We also show that nucleotide excision repair (NER) machinery may counteract the formation of O2-PHBdT and O4-PHBdT, and O6-alkylguanine DNA alkyltransferase (AGT) may be responsible for the repair of O6-PHBdG and O4-PHBdT in mammalian cells. Together, our study provides new knowledge about the occurrence and repair of NNAL-induced DNA lesions in mammalian cells.

Graphical Abstract

graphic file with name nihms-1018807-f0001.jpg

INTRODUCTION

The human genome is susceptible to damage by endogenous metabolites and exogenous chemicals.1,2 The resulting DNA damage leads to perturbations of genomic stability, which may give rise to mutagenesis and other adverse biological consequences.3 Tobacco-specific nitrosamines may contribute to human cancer, and they are associated with the elevated lung cancer rate in the cohorts of active tobacco users in several epidemiological studies.4–6 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a major tobacco-specific nitrosamine, and its reduced metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) have been found to induce cancer in rodents in pioneering studies by Hecht and co-workers.7–9 Carcinogenesis from exposure to NNK and NNAL arises after they give rise to adducts on the nucleobases and backbone of DNA, which in turn results in compromised DNA replication and leads to mutations in DNA.10,11

Some earlier studies by Hecht and others demonstrated that a subset of cytochrome P450 enzymes participate in activation of NNK and NNAL,11–14 which as a result gives rise to reactive intermediates that can pyridyloxobutylate and pyridylhydroxybutylate DNA, respectively (Scheme 1). A number of studies showed the presence of DNA lesions in mammalian genome due to pyridyloxobutylation and pyridylhydroxybutylation. These lesions include O2 -POBdT,15–17 O4 -POBdT,18 O6 -POBdG,15,17,19 O2-PHBdT,15 O6-PHBdG,15,17 O2-POBdC,17 7-POBG,15,17 7-PHBG,15 N6-POBdA,20 N6-PHBdA,20 N1-POBdI,20 and N1-PHBdI20 on nucleobases and B1p(POB)-B221–23 and B1p(PHB)B221–23 on phosphate backbone. Most lesions mentioned above exhibited cytotoxic and mutagenic properties, which, if not properly repaired, may impede the transmission of genetic information by disrupting DNA replication and transcription machineries. Du et al.24 demonstrated recently that O2-POBdT and O4-POBdT could moderately block DNA replication, which elicited T → A transversion and T → C transition, respectively, whereas G → A transition constituted the major form of mutation induced by O6-POBdG. The activated form of NNK and NNAL may also serve as a potent methylating agent. The resulting level of O6-methylguanine (O6-mG) following NNK exposure was found to be correlated with tumorgenicity.19,25 Ma et al.26 also demonstrated recently that B1pmeB2 can be induced in vivo upon exposure to NNK and NNAL.

Scheme 1.

Scheme 1.

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Activation of NNALOAc by Cellular Esterases, and Metabolic Activation of NNK and NNAL by Cytochrome P450 Enzymes, Followed by the Conjugation of the Resulting Common Reactive Intermediate with DNA to Yield O2-PHBdT, O4-PHBdT, and O6-PHBdGa

Mammalian cells contain a complex arsenal of DNA repair proteins to remove deleterious DNA lesions.1,2,27 In this vein, various classes of mammalian DNA repair proteins are capable of removing pyridyloxobutylated and pyridylhydroxybutylated DNA-modifications. O6-alkylguanine-DNA alkyltransferase (AGT) was found capable of removing directly the POB remnant from O6-POBdG.17,19,28 There are also studies revealing that O2-POBdT contributes to the elevated occurrence of AT → TA mutation in cells deficient in nucleotide excision repair (NER); therefore, NER is likely an important pathway for the repair of this lesion.28

Although the implications of tobacco-specific nitrosamines in carcinogenesis have been well documented,14,29,30 there are still gaps to be filled in establishing reliable quantification methods to meet the increasing demands for assessing the risk of tobacco consumption. In this respect, methods including 32P-postlabeling assay, excision assay, and immunoblot analysis have been used for measuring the levels and assessing the repair of targeted DNA lesions in vitro and in vivo.31,32 Due to its high sensitivity and its capacity in providing structural information,18,33–41 mass spectrometry has become a tool of choice in DNA adduct analysis. In this vein, solid-phase extraction followed by LC-MS/MS analysis was previously used for quantifying several pyridyloxobutyl and pyridylhydroxybutyl DNA lesions.17,21,23,26,28

Herein, we report the application of a highly sensitive nanoflow liquid chromatography-nanoelectrospray ionization tandem mass spectrometry (nLC-nESI-MS/MS) coupled with the stable isotope-dilution technique for the simultaneous quantifications of O4-PHBdT, O2-PHBdT, and O6-PHBdG. This is the first reported measurement of O4-PHBdT in mammalian cells (Scheme 1). By employing this method, we further explored how the three lesions are repaired in cultured mammalian cells.

EXPERIMENTAL SECTION

Materials.

Unless further specified, all chemicals were obtained from Sigma-Aldrich (St. Louis, MO), and enzymes were obtained from New England Biolabs (Ipswich, WA). NNALOAc was obtained by the reduction of NNKOAc (Toronto Research Chemicals Inc., North York, Ontario) using NaBH4. The reaction was carried out under a condition where NNKOAc (10 mg) was mixed with 5 equiv of NaBH4 in methanol at room temperature for 30 min, and the product NNALOAc was then isolated from the reaction mixture using HPLC. Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) hydrochloride was obtained from Tocris Bioscience (Ellisville, MO). Repair-competent and ERCC1-deficient AA8 Chinese hamster ovary (CHO and ERCC1, CHO-7−27)42 cells were provided by M. M. Seidman (National Institute of Aging, Bethesda, MD). Human skin fibroblasts with repair proficiency (GM00637) and deficiency in xeroderma pigmentosum (XPA, GM04429) were kind gifts from G. P. Pfeifer (Van Andel Research Institute, Grand Rapids, MI).

Preparation of Standards.

O2-PHBdT, O6-PHBdG, O4-PHBdT, and the respective stable isotope-labeled derivatives were obtained by reduction with NaBH4 of previously synthesized O2-POBdT, O6-POBdG, and O4-POBdT.18 Unlabeled and stable isotope-labeled O4-PHBdT have not been previously synthesized and were made specifically for this study. The reaction was carried out by mixing O4-POBdT (100 μg) with 5 equiv of NaBH4 in 100 μL methanol at room temperature for 10 min. The solution was diluted with 9 volumes of water, and the mixture was subjected to extraction with an equal volume of ethyl acetate for three times. The aqueous layer was collected, and the overall yield was 80%. Exact mass measurements (Thermo Q-Exactive Plus) yielded m/z 392.1820 and 396.2076 for the [M + H]+ ions of the O4-PHBdT and [pyridine-d4]-O4-PHBdT, respectively, which are in agreement with their respective calculated m/z values of 392.1822 and 396.2073 (Table S1, Figure S1). O2-PHBdT and O6-PHBdG were prepared following similar procedures and characterized similarly (Table S1, Figures S2−S3).

Treatment of Calf Thymus DNA with NNALOAc and Porcine Liver Esterase.

Calf thymus DNA (25 μg) was treated with 10 or 50 μg NNALOAc and porcine liver esterase (0.4 U) in 0.1 M phosphate buffer (1 mL, pH 7.0) at 37 °C overnight. The resulting mixture was first extracted with an equal-volume mixture of CHCl3/isoamyl alcohol (24:1, v/v), followed by ethyl acetate extraction. The DNA was precipitated from the aqueous phase by adding cold ethanol, washed sequentially with 70% ethanol and then with 100% ethanol, allowed to air-dry at room temperature, redissolved in doubly distilled water, and kept at−20 °C until enzymatic digestion and subsequent LC-MS/MS analysis.

Cell Culture and Treatment with NNALOAc.

All cells were cultured at 37 °C in a 5% CO2 atmosphere with media containing 10% fetal bovine serum and 100 IU/mL penicillin: Human skin fibroblasts were grown in Dulbecco’s modified Eagle’s medium (Gibco), and CHO cells were grown in Alpha Minimum Essential Medium (Gibco) without ribonucleosides or 2′-deoxyribonucleosides. Cells were seeded (approximately 1−1.5 × 106) in 75 cm2 flasks and cultured for 24 h. The cells were unexposed or exposed with 5, 10, or 25 μM of NNALOAc. The GM00637 human skin fibroblast cells were treated with 10 μM NNALOAc alone or concurrently with 20 μM O6-benzylguanine, an AGT inhibitor. After a 24 h treatment, the medium was removed from the flask, and the cells were washed twice with phosphate-buffered saline (1 × PBS) to remove any remaining NNALOAc. During repair studies, cells were maintained at 37 °C in their respective fresh media, detached using trypsin-EDTA at different time intervals and centrifuged into a cell pellet.

DNA Extraction and Enzymatic Digestion.

Standard procedures for DNA extraction, enzymatic digestion, and HPLC enrichment were utilized for the targeted pyridylhydroxybutylated nucleosides and are detailed previously.18,36,38 A representative chromatogram for off-line HPLC enrichment is shown in Figure S4, and the detailed procedures are provided in the Supporting Information.

nLC-nESI-MS/MS Analysis.

Online nLC-nESI-MS/MS measurements were conducted on a TSQ-Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific, CA) equipped with a nanoelectrospray ionization source and coupled with an EASY nLC II system (Thermo Fisher Scientific, CA). HPLC separation was conducted by employing a trapping column (150 μm × 40 mm) and an analytical column (75 μm × 200 mm), both packed in-house with Magic C18 AQ (200 Å, 5 μm, Michrom BioResource, Auburn, CA). Mobile phases A and B were 0.1% formic acid in doubly distilled H2O and acetonitrile, respectively. Initially, the sample was loaded, at a flow rate of 3 μL/min for 5 min, onto the trapping column with mobile phase A. The modified nucleosides were eluted through a 40 min linear gradient of 0−50% mobile phase B with a flow rate of 300 nL/min. The TSQ-Vantage mass spectrometer was operated in the selected-reaction monitoring (SRM) mode. We monitored the transitions corresponding to the neutral loss of a deoxyribose (116 Da) from the [M + H]+ ions of the three modified nucleosides (i.e., m/z 392 → 276, 392 → 276, and 417 → 301 for O2-PHBdT, O4-PHBdT, and O6-PHBdG, respectively) and their stable isotope-labeled counterparts (i.e., m/z 396 → 280, 396 → 280, and 421 → 305, Figure 1). The voltage for electrospray was set at 2.0 kV, and the ion transfer tube was heated to 275 °C. The widths for precursor ion and product ion isolation were 3 and 0.7 Da, respectively, with a dwell time for 1.25 s. The collision gas was 1.2 mTorr argon, and the collision energy was 15 V. The limit of quantitation (LOQ) here is defined as the amount of analyte needed to yield a signal that is the sum of the mean signal and 10 times the standard deviation of signal from three blank runs in the selected-ion chromatograms (SICs) plotted for the transitions used for analyte quantification.

Figure 1.

Figure 1.

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Representative SICs for the m/z 392 → 276 (A, top panel), 396 → 280 (A, bottom panel), 417 → 301 (B, top panel), and 421 → 305 (B, bottom panel) transitions for the [M + H]+ ions of the unlabeled and stable isotope (i.e., pyridine-d4)-labeled O2- and O4-PHBdT (A) and O6-PHBdG (B), respectively, in the enriched modified nucleoside mixture of genomic DNA extracted from the GM04429 cells treated with 10 μM NNALOAc for 24 h.

METHOD DEVELOPMENT

The intra- and interday precision and accuracy of the O2-PHBdT, O4-PHBdT, and O6-PHBdG measurements were gathered from three different concentrations of each analyte. The samples used for calibration curve generation or precision and accuracy test were comprised of 5 μg calf thymus DNA mixed with standard solutions of the three lesion-containing oligodeoxyribonucleotides (ODNs, 5′-ATGGCGXGCTAT-3′, where ‘X’ represents O2-PHBdT, O4-PHBdT, or O6-PHBdG) and their corresponding stable isotope-labeled mononucleosides. The samples from cellular DNA were prepared following previously described procedures 18,36,38 and were subjected to LC-MS/MS measurements. Calibration curves were obtained from triplicate analyses, and the molar ratios of the unlabeled ODNs to their stable isotope-labeled counterparts were 0.10, 0.50, 1.00, 2.00, 5.00, 10.0, and 20.0 for O2-PHBdT and O6-PHBdG, and O4-PHBdT, with the amount of the labeled nucleosides being 5 fmol each. Calibration curves were constructed using SIC peak area ratios for the unlabeled/labeled standards versus the molar ratios of unlabeled/labeled standards and fitting a straight line (Figure S5). The moles of DNA lesions in the nucleoside mixtures were calculated from the peak area ratios found in the SICs for the analytes and compared to their corresponding stable isotope-labeled counterparts. The final levels of DNA lesions were displayed as number of lesions per 108 nucleosides and were calculated by dividing the number of moles of modified by moles of total nucleosides in the digestion mixture.

RESULTS

In this study, we aimed to set up a robust method for the quantification of O-pyridylhydroxybutylated dT and dG lesions in enzymatically digested cellular DNA using nLC-nESI-MS/MS coupled with the stable-isotope-dilution, and we set out to employ the method for examining the occurrence and repair of these lesions in mammalian cells.

Syntheses of Unlabeled and Stable Isotope-Labeled Standards.

We prepared the unlabeled and stable isotope-labeled standards for O2-PHBdT, O4-PHBdT, and O6-PHBdG (see Experimental Section). O4-PHBdT is a novel pyridylhydroxylbutylated DNA lesion that we synthesized through reduction of the previously synthesized O4-POBdT.18

Quantification of O2-PHBdT, O4-PHBdT, and O6-PHBdG Lesion Using nLC-nESI-MS/MS.

We next employed the nLC-nESI-MS/MS method for accurate quantifications of O2-PHBdT, O4-PHBdT, and O6-PHBdG. The LOQs of these three analytes were determined to be 24.0, 19.0, and 8.5 amol, which correspond to 7.4, 5.9, and 2.6 modifications per 1010 nucleosides, respectively, when 10 μg DNA is used for analysis. The procedures for sample preparation are described in detail in the Experimental Section, where the cellular samples were subjected to DNA extraction using a high-salt method, RNA removal, and enzymatic digestion with a cocktail of four enzymes, including both 3′ and 5′ exonucleases to release DNA lesions as mononucleosides. The stable isotope-labeled O2-PHBdT, O4-PHBdT, and O6-PHBdG were added prior to DNA digestion, which corrects for the potential analyte loss during the extraction and HPLC enrichment steps. We then evaluated the intra- and interday precision and accuracy by triplicate measurements of calf thymus DNA (10 μg) mixed with various concentrations of lesion-bearing ODNs. Our results demonstrate that the precision (relative standard deviation: 3.8−14.9%) and accuracy (recovery: 84.9−97.1%) for the method were reasonably good (Table 1).

Table 1.

Intraday and Interday Evaluation on Precision and Accuracy, as Represented by Relative Standard Deviation (RSD) and Recovery, Respectively, for the Measurements of O2-PHBdT, O4-PHBdT, and O6-PHBdG

Intraday
 interday
ODN amount (fmol) expected lesion frequency in 10 μg DNA (/108 nucleosides) RSD (%) recovery (%) RSD (%) recovery (%)
O2-PHBdT
  5.0  15.4  8.1 91.2 14.9 84.9
 15  46.2  5.6 89.3  9.1 90.0
 50 154.0  7.4 90.5 11.6 87.1
O4-PHBdT
  5.0  15.4  9.2 93.4 11.0 90.4
 15  46.2  7.7 88.2 13.9 89.5
 50 154.0 10.1 92.5 10.7 87.8
O6-PHBdG
  5.0  15.4  7.8 95.2  9.5 89.6
 15  46.2  6.1 94.6 14.3 97.1
 50 154.0  3.8 90.7 11.5 91.0
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The detection of DNA modifications of low abundance is challenging, where the sensitivity for analyte measurement is sometimes compromised by much larger amounts of canonical nucleosides released from DNA and buffer salts included in the enzymatic digestion reaction. To address this issue, we conducted offline LC enrichment before LC-MS/MS analysis. The LC-MS/MS conditions were optimized prior to sample analysis. Better sensitivity could be obtained in the positive-ion mode, which was used along with 0.1% formic acid (v/v) in the mobile phase to improve protonation of the analytes.

Dose-Dependent Formation of O2-PHBdT, O4-PHBdT, and O6-PHBdG in Mammalian Cells.

After successfully developing a robust method for nLC-nESI-MS/MS analysis, we next employed the method to quantify the levels of O2-PHBdT, O4-PHBdT, and O6-PHBdG in genomic DNA. The DNA samples were isolated from Chinese hamster ovary cells or human skin fibroblasts exposed with varied concentrations of NNALOAc. NNALOAc was used as a cell membrane-permeable precursor to the reactive intermediate in the presence of cellular esterase (Scheme 1). All three modified nucleosides formed in a dose-dependent manner with increasing concentration of NNALOAc (Figure 2). When the concentration of NNALOAc was increased from 5 to 25 μM, the amount of O4-PHBdT in the XPA-proficient GM00637 cells and XPA-deficient GM04429 cells increased from 5.8 to 33.0 and from 9.5 to 53.1 lesions per 108 nucleosides, respectively (Figure 2B). Similar elevated levels of O4-PHBdT were observed in Chinese hamster ovary cells, where in ERCC1-deficient CHO-7−27 cells and in repair-proficient CHO-AA8 cells, the respective frequencies of O4-PHBdT were 16.4−71.5 and 12.0−47.4 lesions per 108 nucleosides (Figure 2E). Our quantification data also showed that the levels of O2-PHBdT and O6-PHBdG were significantly higher than that of O4-PHBdT in mammalian cells.

Figure 2.

Figure 2.

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Frequencies of O2-PHBdT (A, D), O4-PHBdT (B, E), and O6-PHBdG (C, F) in DNA samples isolated from human skin fibroblast cells (A−C) that are repair proficient (GM00637) or deficient in XPA (GM04429) and Chinese hamster ovary cells (D−F) that are repair competent (CHO-AA8) or deficient in ERCC1 (CHO-7−27). The cells were exposed to the indicated concentrations of NNALOAc for 24 h. The data represent the mean and standard deviation of results obtained from three independent experiments. *, 0.01 ⩽ p < 0.05; **, 0.001 ⩽ p < 0.01; ***, p < 0.001. The p values were calculated by using unpaired two-tailed student’s t-test.

The pronounced difference in occurrence between O2-PHBdT and its regioisomeric O4-PHBdT might arise from the differences in their rate of formation or repair. To explore the former possibility, we exposed calf thymus DNA to NNALOAc together with porcine liver esterase and quantified the frequencies of the three lesions using the aforementioned workflow (Figure 3). Our in vitro results validated the preferential formation O2-PHBdT over O4-PHBdT, while O6-PHBdG formed at higher levels than either of the other two lesions. The levels of O2-PHBdT, O4-PHBdT and O6-PHBdG—relative to one another—were in line with what were previously reported for the corresponding POB adducts in calf thymus DNA treated with NNKOAc and esterase.18,22,39

Figure 3.

Figure 3.

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Frequencies of O4-PHBdT, O2-PHBdT, and O6-PHBdG in calf thymus DNA treated with (A) 10 μg (37.4 μM) and (B) 50 μg (187 μM) NNALOAc in the presence of porcine liver esterase.

Removal of O2-PHBdT, O4-PHBdT, and O6-PHBdG in Mammalian Cells.

Our quantification results reveal that the two lines of NER-deficient cells (i.e., XPA-deficient GM04429 and ERCC1-deficient CHO-7−27) exhibited significantly higher levels of O2-PHBdT and O4-PHBdT compared to their repair-competent counterparts (GM00637 and CHO-AA8). These results substantiate the notion that NER is involved in removal of these two lesions from cellular DNA. To further assess the involvement of NER in repairing these lesions, we also measured the frequencies of the three pyridylhydroxybutyl lesions in the cells at 0, 12, and 24 h after treatment with 10 μM NNALOAc (Figure 4). No pronounced cell death was observed at the end of NNALOAc exposure. Our results showed that the rates of decrease of this specific lesion were comparable in human skin fibroblasts cells and Chinese hamster ovary cells. In addition, the levels of O2-PHBdT in NER-proficient and NER-deficient cells were significantly different at all three time points following NNALOAc exposure, substantiating that O2-PHBdT is a good substrate for NER (Figure 4A,D). To a lesser degree, O4-PHBdT is also affected by NER, as supported by the quantification data obtained for this lesion in NER-deficient cells and their repair-proficient counterparts (Figure 4B,E). O6-PHBdG was found not to be an NER substrate, as the levels of this lesion in NER-deficient cells are not significantly different from that in NER-proficient cells measured at all time points (Figure 4C,F). We also observed that the CHO cells and human skin fibroblasts cells exhibited different repair efficiencies for O6-PHBdG, suggesting that AGT may be involved in repairing this specific lesion, since there is no AGT expression in CHO cells.17,19,28,43 In this vein, it is of note that the time-dependent decrease in the levels of the three lesions is attributed, in part, to the increase in cell numbers over time.

Figure 4.

Figure 4.

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LC-MS/MS for monitoring the repair of O2-PHBdT (A, D), O4-PHBdT (B, E), and O6-PHBdG (C, F) in human skin fibroblast (A−C) and Chinese hamster ovary (D−F) cells. After a 24 h exposure to 10 μM NNALOAc, the media was exchanged, and the cells were harvested immediately or 12 or 24 h later. The data represent the mean and standard deviation of results obtained from three independent experiments. *, 0.01 ⩽ p < 0.05; **, 0.001 ⩽ p < 0.01; ***, p < 0.001. The p values were calculated by using unpaired two-tailed Student’s t-test.

We also asked whether AGT participated in the removal of the three lesions. Toward this end, we exposed GM00637 cells with 10 μM NNALOAc alone, or in conjunction with 20 μM O6-benzylguanine, and measured the levels of the three lesions at 24 h after exposure. Along this line, it was observed previously that treatment of cultured mammalian cells with 20 μM O6-benzylguanine could result in the ubiquitination and proteasomal degradation of AGT protein, where the protein is almost undetectable at 18 h following the treatment.44 It turned out that the levels of O2-PHBdT in the control and AGT-depleted cells were almost the same (Figure 5A). We detected a small difference in the levels of O4-PHBdT in GM00637 cells with or without O6-benzylguanine treatment (Figure 5B), indicating that AGT may also be involved in the removal of O4-PHBdT.27,45 The difference in O6-PHBdG levels was the largest among the three lesions (Figure 5C), suggesting that AGT readily repairs this lesion.

Figure 5.

Figure 5.

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Repair of O2-PHBdT (A), O4-PHBdT (B), and O6-PHBdG (C) in GM00637 human skin fibroblast cells following a 24 h exposure to 10 μM NNALOAc alone or concurrently with 20 μM O6-benzylguanine (to deplete AGT). The data represent the mean and standard deviation of results obtained from three independent experiments. *, 0.01 ⩽ p < 0.05; **, p < 0.01. The p values were calculated by using unpaired two-tailed Student’s t-test.

DISCUSSION

LC-MS/MS in combination with the stable isotope-dilution method enables specific, accurate, and highly sensitive measurement of DNA lesions in complex biological matrices.18,33–41 It is advantageous over conventional DNA adduct measurement methods (e.g., immunoblot assay, 32P-postlabeling)46–48 by offering structural information to enable identification and by facilitating reliable absolute quantification when spiked with known amounts of stable isotope-labeled standards.40,49,50 In this study, stable isotope-labeled standards were added prior to enzymatic digestion, which helps correct for the loss of analytes during chloroform extraction and offline enrichment; thereby providing more accurate quantification for the three lesions. Additionally, offline HPLC was employed to eliminate the more abundant canonical nucleosides in digestion mixtures and remove buffer salts used during the enzymatic digestion. Our offline HPLC enrichment method provides higher sensitivity in subsequent LC-MS/MS analysis compared to previously reported solid-phase extraction methods for targeted nucleoside enrichment; this lower sensitivity may have been due to incomplete removal of unmodified nucleosides and buffer salts during solid-phase extraction.51,52 The calibration curve established in this study employed calf thymus DNA spiked with lesion-bearing ODNs, which allowed us to correct for potential incomplete release of modified nucleosides from DNA during the digestion step.

Mammalian cells contain an arsenal of DNA repair proteins that can be dispatched in response to numerous types of damage, thus protecting genomic stability. Here we used four lines of mammalian cells that are proficient or deficient in key NER proteins, and we investigated the role of NER in the removal of O2-PHBdT, O4-PHBdT, and O6-PHBdG from genomic DNA. Dose-dependent formation was observed for O2-PHBdT, O4-PHBdT, and O6-PHBdG in the four cell lines, where O2-PHBdT and O6-PHBdG displayed higher frequencies of formation than O4-PHBdT. In the repair study, we carefully compared the levels of these lesions in NER-proficient and NER-deficient cells. Our results revealed that O2-PHBdT and, to a lesser extent, O4-PHBdT may serve as substrates for NER, whereas O6-PHBdG cannot be repaired by NER. The different capacity of Chinese hamster ovary cells and human fibroblasts cells in repairing O6-PHBdG suggests that AGT is responsible for the removal of this specific modification.17,19,28,43 Thus, we interrogated how the accumulation of the three lesions in human cells is affected by co-exposure with an AGT inhibitor, O6-benzylguanine. The results confirmed our hypothesis that AGT assumes an important role in the reversal of O6-PHBdG and, to a lesser degree, O4-PHBdT, which is in line with the notion that AGT recognizes major-groove lesions.27,45

It is worth comparing the results obtained for the pyridylhydroxybutylated lesions with those obtained previously for the pyridyloxobutylated lesions.18 In this context, our previous study on O2-POBdT, O4-POBdT, and O6-POBdG revealed that the formation of the three POB lesions is dose-dependent, and O2-POBdT is most efficiently repaired by NER, O4-POBdT can be repaired at a lesser degree, whereas O6-POBdG is not a substrate for NER.18 The involvement of AGT in repairing O6-PHBdG is in keeping with our previous observation of lower levels of O6-POBdG in human skin fibroblasts than CHO cells.18

In summary, here we reported an LC-MS/MS method for the simultaneous measurement of three pyridylhydroxybutyl lesions, that is, O2-PHBdT, O4-PHBdT, and O6-PHBdG, where O4-PHBdT was, for the first time, found to be induced in mammalian cells upon exposure to pyridylhydroxybutylating agents. The method exhibited high sensitivity in quantification of targeted DNA lesions and its capacity to snapshot the levels of lesions in a repair study at different time intervals. The method may serve as a tool for investigating the involvement of DNA pyridylhydroxybutylation as a biomarker related to tobacco-induced cancer. In this regard, it will be important to examine, in the future, the occurrence and repair of these lesions in human lung cells and the lung tissues of smokers.

Supplementary Material

Supplementary Materials

ACKNOWLEDGMENTS

This work was supported by the National Institute of Health (R01 ES025121), and N.P. was supported in part by an T32 Institutional Training Grant (ES018827).

Footnotes

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00374.

Figures S1−S3 (ESI-MS and MS/MS of O4-PHBdT, O2-PHBdT, and O6-PHBdG and their stable isotope-labeled derivatives); Figure S4 (HPLC trace for the enrichment of O4-PHBdT, O2-PHBdT, and O6-PHBdG from the enzymatic digested monomers of genomic DNA isolated from NNALOAc-treated cells); Figure S5 (calibration curves for the quantifications of O4-PHBdT, O2-PHBdT, and O6-PHBdG). Table S1 (accurate mass measurement results for the [M + H]+ ions of unlabeled and the stable isotope-labeled O4-PHBdT, O2-PHBdT, and O6-PHBdG) (PDF)

Notes

The authors declare no competing financial interest.

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