Methyl DNA phosphate adduct formation in lung tumor tissue and adjacent normal tissue of lung cancer patients

Bin Ma; Peter W Villalta; J Bradley Hochalter; Irina Stepanov; Stephen S Hecht

doi:10.1093/carcin/bgz053

. 2019 Mar 12;40(11):1387–1394. doi: 10.1093/carcin/bgz053

Methyl DNA phosphate adduct formation in lung tumor tissue and adjacent normal tissue of lung cancer patients

Bin Ma ^1,^✉, Peter W Villalta ¹, J Bradley Hochalter ¹, Irina Stepanov ¹, Stephen S Hecht ¹

PMCID: PMC6875899 PMID: 30873516

Abstract

The formation of methyl DNA adducts is a critical step in carcinogenesis initiated by the exposure to methylating carcinogens. Methyl DNA phosphate adducts, formed by methylation of the oxygen atoms of the DNA phosphate backbone, have been detected in animals treated with methylating carcinogens. However, detection of these adducts in human tissues has not been reported. We developed an ultrasensitive liquid chromatography–nanoelectrospray ionization–high resolution tandem mass spectrometry method for detecting methyl DNA phosphate adducts. Using 50 μg of human lung DNA, a limit of quantitation of two adducts/10¹⁰ nucleobases was achieved. Twenty-two structurally unique methyl DNA phosphate adducts were detected in human lung DNA. The adduct levels were measured in both tumor and adjacent normal tissues from 30 patients with lung cancer, including 13 current smokers and 17 current non-smokers, as confirmed by measurements of urinary cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Levels of total methyl DNA phosphate adducts in normal lung tissues were higher in smokers than non-smokers, with an average of 13 and 8 adducts/10⁹ nucleobases, respectively. Methyl DNA phosphate adducts were also detected in lung tissues from untreated rats with steady-state levels of 5–7 adducts/10⁹ nucleobases over a period of 70 weeks. This is the first study to report the detection of methyl DNA phosphate adducts in human lung tissues. The results provide new insights toward using these DNA adducts as potential biomarkers to study human exposure to environmental methylating carcinogens.

Using an ultrasensitive high-resolution tandem mass spectrometry method, methyl DNA phosphate adducts were detected for the first time in human lung tissues. These DNA adducts could serve as biomarkers to study human exposure to environmental and lifestyle methylating carcinogens.

Introduction

DNA adducts, formed either by reaction of carcinogens or their metabolites with DNA, can cause miscoding and mutations. If these occur in critical genes such as p53 and KRAS, the result can be loss of normal growth control processes and cancer (1). Consequently, DNA adducts are considered as potential biomarkers to investigate cancer susceptibility on exposure to carcinogens. Methyl DNA adducts are formed by a number of environmental methylating agents including tobacco carcinogens such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (2–4), and pesticides such as methyl carbamates and methyl parathion (5). Methyl DNA adducts are also formed endogenously by S-adenosylmethionine and probably from other compounds such as betaine and choline (6,7). Medicinal exposure to methylating drugs such as dacarbazine and temozolomide is another source of exposure for patients undergoing chemotherapy (8,9).

Methyl DNA base adducts, formed by the reaction of methylating species with A, G, C or T in DNA, have been detected in both animal models and humans (2–6). Methyl DNA phosphate adducts can be produced by reaction of methylating agents with the DNA phosphate backbone (3). For methyl base adducts, 7-methyl-2′-deoxyguanosine (7-mdG) is generally the most abundant one, but 7-mdG itself does not miscode or block replication (10). Instead, its ring-opened fapy adduct blocks replication and can be mutagenic (11). Also, the abasic sites resulting from depurination of 7-mdG are mutagenic and toxic (12). The major mutagenic and toxic methyl base adduct is O⁶-methyl-2′-deoxyguanosine (O⁶-mdG), which induces primarily GC→AT transition mutations. It can also trigger apoptosis if not repaired (13–15). Some other minor methyl base adducts also demonstrated mutagenicity and cytotoxicity (14). Compared with methyl base adducts, the biological significance of methyl DNA phosphate adducts is currently understudied. Previous studies suggested some effects of methyl DNA phosphate adducts, including inhibiting DNA metabolic enzymes and helicases and interfering with the binding of DNA to protein (16–18). A recent study demonstrated that thymine-containing methyl DNA phosphate adducts can cause TT→GT and TT→GC mutations in Escherichia coli cells (19). The same study also showed that (S) isomers of methyl DNA phosphate adducts were efficiently bypassed by DNA polymerases, whereas the (R) isomers moderately blocked DNA replication in E.coli. However, the overall biological role of methyl DNA phosphate adducts remains largely unknown.

One major challenge of analyzing methyl DNA phosphate adducts is the complexity of their chemical structures. After the DNA is enzymatically hydrolyzed, the methyl DNA phosphate adducts are measured as methyl phosphotriesters—B₁pMeB₂ (Figure 1) (3). There are two nucleobases in the structure, which can have 10 base combinations depending on which two bases comprise the adduct. Because of the chiral center in the phosphate group, there can be (R) and (S) isomers with the same bases and four isomers if the two bases are different, which results in a total of 32 possible isomers (Table 1) (3). We have developed a novel liquid chromatography (LC)–nanoelectrospray ionization (NSI)–high-resolution tandem mass spectrometry (HRMS/MS)-based method for the analysis of methyl DNA phosphate adducts in NNK- and NNAL-treated rats (3). A total of 23 of 32 methyl DNA phosphate adduct isomers were detected in that study and the adduct levels were quantified in the lung tissues of those treated rats. However, the methyl DNA phosphate adducts were not detected in rats from the control group without carcinogen treatment (3), even though the rats were possibly exposed to trace levels of methylating agents present in their diet and formed endogenously. Similarly, the methyl DNA phosphate adducts in humans may also exist at extremely low levels due to the fact that environmental methylating agents are generally present at trace levels. To date, the detection of methyl DNA phosphate adducts in human tissues has to our knowledge not been reported.

Figure 1. — Analytical scheme for the analysis of methyl DNA phosphate adducts (B₁pMeB₂) in human and rat lung tissues. B₁ and B₂ represent the same or different nucleobases.

Table 1.

Methyl DNA phosphate adducts detected in human and rat lung DNA samples

B₁pMeB₂	[M + H]⁺	Scan range (min)	Possible	Number of isomers detected
				Rat	Human
					Normal tissue	Tumor tissue
A-A	579.1824	28.4–32.6	2	1	2	2
C-C	531.1599	0–25.0	2	1	2	2
G-G	611.1722	0–25.0	2	1	1	1
T-T	561.1592	32.6–53.0	2	2	2	2
A-C	555.1711	0–28.4	4	2	3	2
A-G	595.1773	25.0–28.4	4	2	2	2
A-T	570.1708	28.4–53.0	4	2	2	2
C-G	571.1661	0–25.0	4	2	2	2
C-T	546.1596	25.0–32.6	4	3	3	3
G-T	586.1657	25.0–32.6	4	2	3	3
Total			32	18	22	21

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In this study, we modified the previous LC–NSI–HRMS/MS method by optimizing the sample preparation protocol and mass spectrometry parameters to improve the detection sensitivity. Using the optimized method, we measured methyl DNA phosphate adducts in both lung tumor tissue and the adjacent normal tissue of patients with lung cancer. A unique feature of this study was confirmation of smoking status at the time of lung cancer surgery by measurement of urinary cotinine and NNAL. We also applied our optimized LC–NSI–HRMS/MS method to the analysis of methyl DNA phosphate adducts in lung tissues from the untreated rats in our previous study. Our ultimate goal was the application of this optimized method to study environmental and medicinal exposure to methylating agents and the biological consequences of methyl DNA phosphate adduct formation.

Materials and methods

Materials and chemicals

Thymidylyl(3′-5′)thymidine methyl phosphotriester (TpMeT) was purchased from MRIGlobal (Kansas City, MO). [¹³C₁₀¹⁵N₂]TpMeT was available from our previous study (3). Enzymes and reagents for DNA isolation were purchased from QIAGEN Sciences (Germantown, MD). Deoxyribonuclease I (from bovine pancreas) and phosphodiesterase I (from Crotalus adamanteus venom) were purchased from Sigma–Aldrich (Milwaukee, WI). Alkaline phosphatase (from calf intestine) was obtained from Roche (Indianapolis, IN). All other chemicals and solvents were obtained from Sigma–Aldrich.

Tissue samples

Human lung tissue samples were from the University of Minnesota Medical School Tissue Procurement Facility. After informed consent, tumor tissue samples were obtained during surgery for lung cancer, and the tissue samples from the margins of tumors were also collected and represent pathologically normal lung surrounding tumor tissue. Urine samples from the same individuals were obtained before surgery. This study was approved by the University of Minnesota Institutional Review Board (IRB #0305M47681). Lung tissue samples of untreated F-344 rats (n = 3) at age 10, 30, 50 and 70 weeks were from a previous study, in which those rats were in the control group (2).

Analysis of urine for total cotinine and total NNAL

To confirm the self-reported smoking status of the patients with lung cancer, their urine samples were analyzed for total cotinine (cotinine plus its glucuronide) and total NNAL (NNAL plus its two glucuronides), which are widely used biomarkers of exposure to cigarette smoking (20). The samples were analyzed as described previously (21,22), and the levels of total cotinine and total NNAL are presented in Supplementary Table 1, available at Carcinogenesis Online.

DNA hydrolysis and sample preparation

DNA was extracted from lung tissues using our previously developed protocol (23). The DNA samples were then hydrolyzed and purified by using our previously developed protocol with modifications (3). One modification was purification of the DNA hydrolysis enzymes by multiple centrifugal filtration steps before use to remove salts and other impurities following our previously developed protocol (24). To start the DNA hydrolysis, the DNA samples (~50 µg) were dissolved in 0.5 ml of 5 mM sodium succinate (pH 7.4) buffer containing 2 mM CaCl₂ and 5 fmol [¹³C₁₀¹⁵N₂]TpMeT as internal standard, followed by adding a cocktail of purified enzymes, including deoxyribonuclease I (0.75 units), phosphodiesterase I (0.005 units) and alkaline phosphatase (0.4 units). After incubating at 37°C for 16 h, 20 μl hydrolysate was collected for DNA quantitation by analyzing the level of deoxyguanosine (23), which accounts for 21% of the total nucleotides. The amount of DNA was then calculated from the deoxyguanosine content by considering that 1 mg of DNA contains 3 μmol of nucleotides (23,25). The remaining hydrolysates were filtered with 10K centrifugal filters (Ultracel YM-10, Millipore), and the filtrates were loaded on 30 mg Strata-X cartridges (33 μm polymeric reversed phase, 30 mg; Phenomenex) activated with 3 ml MeOH and 3 ml H₂O. The cartridges were washed with 2 ml H₂O and 1 ml of 10% MeOH sequentially and finally eluted with 2 ml of 50% MeOH. The 50% MeOH fraction containing the methyl DNA phosphate adducts was concentrated to dryness in a centrifugal evaporator. The residue was reconstituted in 6 μl of deionized H₂O before analysis by LC–NSI–HRMS/MS.

LC–NSI–HRMS/MS analysis

The analysis was performed on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, Waltham, MA). A nanoLC column (50 μm i.d., 360 μm o.d., 20 cm length) packed with Luna C₁₈ bonded separation media (Phenomenex, Torrance, CA) was used. The mobile phase consisted of 2 mM NH₄OAc and CH₃CN. A 5 μl injection loop was used and the sample (3 μl) was loaded onto the capillary column at 600 nl/min at 2% CH₃CN, and after 9 min the injection valve position was switched to take the injection loop out of the flow path and the flow rate was reduced to 150 nl/min. Separation on the column was performed using a linear gradient with increasing CH₃CN from 2% to 15% for 30 min, followed by ramping to 90% CH₃CN in 2 min and holding at this composition for an additional 3 min. The gradient was then returned to 2% CH₃CN in 1 min, and the system was re-equilibrated for 9 min at 600 nl/min before the next injection. The ion source spray voltage was 2.2 kV, the capillary temperature was 300°C, and the S-Lens RF Level was 60%. The [M + H]⁺ ions of all possible phosphate adducts listed in Table 1 were monitored using selected-ion monitoring mode at different time ranges based on their individual retention times (Table 1). The ions were analyzed using an isolation window of 1.5 m/z, automatic gain control target of 2×10⁵, maximum inject time of 1000 ms and a resolution of 240 000. The product ion scan was performed using higher-energy collisional dissociation fragmentation with a normalized collision energy of 20 units, isolation widths of 1.5 Da for all the precursor ions, and product ion analysis performed with a mass range of m/z 100–700 at a resolution of 15 000. The accurate mass tolerance used for extraction of precursor and fragment ion signals was 5 ppm.

The quantitative analysis was performed as described previously (3). All data are presented as mean ± standard deviation. Two-tailed unpaired t-test was used for two-group comparison, and one-way analysis of variance followed by a Bonferroni post-test was used for multiple comparisons. A P value < 0.05 was considered significant.

Results

Method sensitivity optimization

We previously developed an LC–NSI–HRMS/MS method for the analysis of methyl DNA phosphate adducts in NNK- or NNAL-treated rats (3). The sensitivity of this method was improved here by addressing several aspects based on our experience optimizing a methodology for the detection of the BPDE-N²-dG DNA adduct (24). First, the enzymes used for DNA hydrolysis were purified before use. On the basis of the sources (see details in Materials and chemicals) and isolation processes of the enzymes, there are salts, contaminating small molecules and impurities present which can affect mass spectrometry sensitivity. Washing the enzymes by multiple centrifugal filtration steps has been shown to be an effective purification method to reduce matrix effects resulting from DNA hydrolysis enzymes (24,26). Second, the flow rate during the analysis was reduced from 300 nl/min to 150 nl/min with the corresponding reduction in column internal diameter from 75 to 50 µm i.d., which resulted in a 2- to 3-fold increase in ion signal of the standard [¹³C₁₀¹⁵N₂]TpMeT (Supplementary Figure 1, available at Carcinogenesis Online). Third, a maximum injection time of 1000 ms (100 ms in the previous method) was used to maximize the ions in the Orbitrap detector, leading to increased sensitivity. With such a long injection time, segment scanning was used to monitor different precursor ions of methyl DNA phosphate adducts based on their retention times (Table 1), which allowed maintenance of a sufficient number of chromatographic data points for quantitation. The advantage of using maximum injection time was presented in detail in our previous study (24). Finally, the sample residue was reconstituted in a smaller volume of 6 µl (15 µl previously) while keeping the injection volume constant. With all the optimized parameters in the current method, we were able to detect methyl DNA phosphate adducts in lung samples from both humans and untreated rats. An analytical scheme of the current method is outlined in Figure 1.

Adduct formation in human lung tissue

Using the optimized LC–NSI–HRMS/MS method, a limit of detection of 4 amol (10^–18 mol) on-column was achieved for the standard TpMeT. With ~50 µg human lung DNA, certain methyl DNA phosphate adducts such as GpMeG were detected as low as 0.71 ± 0.52 fmol/mg DNA (Supplementary Table 2, available at Carcinogenesis Online), which is equivalent to two adducts/10¹⁰ bases. At least one isomer of each base combination was observed and a total of 22 and 21 isomers of methyl DNA phosphate adducts were detected in normal and tumor lung tissue samples, respectively (Table 1). A typical chromatogram obtained on the analysis of TpMeT in the normal lung tissue DNA from a smoker is presented in Figure 2A, and the identities of TpMeT and [¹³C₁₀¹⁵N₂]TpMeT were confirmed by comparing with the standards. A chromatogram of CpMeT from the same sample is shown in Figure 2B. The identities of the three peaks were confirmed to be isomers of CpMeT by accurate mass of its precursor ion and matched chromatograms of its major fragment ions.

Figure 2. — Chromatograms of (A) TpMeT and (B) CpMeT in the DNA of a normal lung tissue sample from a smoker. SIM, selected-ion monitoring.

The smoking status of the 30 patients was confirmed by measuring the levels of total cotinine and total NNAL in their urine samples collected when the surgery was conducted. As shown in Supplementary Table S1, available at Carcinogenesis Online, these smokers had 647−18 500 ng/ml total cotinine and 0.48−26 pmol/ml total NNAL, confirming that they smoked virtually until their day of surgery, whereas total cotinine and total NNAL were very low or below the limit of quantitation in the non-smokers. The levels of methyl DNA phosphate adducts were measured in both normal and tumor lung tissues from 13 smokers and 17 non-smokers (Figure 3A and B). In smokers, the levels of total methyl DNA phosphate adducts in normal and tumor tissues were 13−71 fmol/mg DNA, or 4−24 adducts/10⁹ bases and 4−54 fmol/mg DNA, or 1−18 adducts/10⁹ bases, respectively. Twelve patients had higher levels of total methyl DNA phosphate adducts in normal tissues than tumor tissues, whereas only one patient had higher adduct levels in the tumor tissue (Figure 3A). Overall, the levels of total methyl DNA phosphate adducts in normal tissues were 2-fold higher (38 ± 18 versus 20 ± 13 fmol/mg DNA, P < 0.05) than in tumor tissues (Figure 3C). Some of the individual base combinations of the adducts also demonstrated higher levels in normal tissues compared with tumor tissues. For example, the levels of ApMeG in normal tissues were 3-fold higher (3.5 ± 2.7 versus 1.3 ± 1.3 fmol/mg DNA, P < 0.05) than in tumor tissues (Supplementary Table 2A, available at Carcinogenesis Online). In non-smokers, the levels of total methyl DNA phosphate adducts in normal and tumor tissues were 8−45 fmol/mg DNA, or 3−15 adducts/10⁹ bases and 5−74 fmol/mg DNA, or 2−25 adducts/10⁹ bases, respectively. Nine patients had higher levels of total methyl DNA phosphate adducts in normal tissues than tumor tissues, whereas the other eight patients had higher adduct levels in the tumor tissues (Figure 3B). No difference of adduct levels was observed between normal and tumor tissues (23 ± 11 versus 23 ± 15 fmol/mg DNA) of the overall 17 non-smokers (Figure 3D). The adduct levels of individual base combinations between normal and tumor tissues also showed no difference (Supplementary Table 2B, available at Carcinogenesis Online).

Figure 3. — Levels of total methyl DNA phosphate adducts in normal and tumor lung tissues from (A) smokers and (B) non-smokers; comparison of total methyl DNA phosphate adduct levels between normal and tumor tissues from (C) smokers, (D) non-smokers, and between smokers’ and non-smokers’ normal (E) and tumor (F) lung tissues. The median values of DNA adductlevels are indicated by the lines inside the box of (C), (D), (E), and (F). *P < 0.05, two-tailed paired t-test; ^#P < 0.05, two-tailed unpaired t-test.

When comparing the total adduct levels in normal tissues between smokers and non-smokers, a significant difference was observed, with 38 ± 18 fmol/mg DNA in smokers compared with 23 ± 11 fmol/mg DNA in non-smokers (P < 0.05; Figure 3E). Differences were also observed for certain individual base combinations. For example, the levels of ApMeC in smokers were 3-fold higher (7.1 ± 7.9 versus 2.1 ± 1.6 fmol/mg DNA, P < 0.05) than in non-smokers (Supplementary Table 2C, available at Carcinogenesis Online). No difference was observed between smokers and non-smokers in their tumor tissues (Figure 3F), except that the levels of CpMeC were lower in smokers compared with non-smokers (Supplementary Table 2D, available at Carcinogenesis Online).

Adduct formation in rat lung tissue

Methyl DNA phosphate adducts were not detected in the untreated rats in our previous study (3). In this study, we were able to detect these adducts in the same rat lung DNA samples using the optimized method with improved sensitivity. At least one isomer of each base combination was observed, and a total of 18 isomers of methyl DNA phosphate adducts were detected (Table 1). The levels of total methyl DNA phosphate adducts were 10−22 fmol/mg DNA, or 3−7 adducts/10⁹ bases, and the levels were not significantly changed over 10−70 weeks (Figure 4).

Figure 4. — Levels of methyl DNA phosphate adducts in lung DNA of untreated rats at 10, 30, 50 or 70 weeks. Values are presented as means ± SD (n =3).

The levels of methyl DNA phosphate adducts observed in the lung tissues of untreated rats were comparable with the levels in the lung tissues of non-smokers but lower than smokers (Table 2). With the treatment of the methylating agent NNK, the adduct levels increased significantly. For example, the total adduct levels increased by 300-fold in NNK-treated rats compared with the untreated rats at 30 weeks (Table 2).

Table 2.

Levels of total methyl DNA phosphate adducts in lung tissues from humans, untreated rats and NNK-treated rats

Lung tissue	Total methyl DNA phosphate adducts (adducts/10⁹ bases)
Normal tissue
Smokers (n = 13)	13 ± 6
Non-smokers (n = 17)	8 ± 4
Untreated rats (n = 3)
10 weeks	6 ± 0.2
30 weeks	5 ± 1
50 weeks	5 ± 1
70 weeks	7 ± 1
NNK-treated rats (n = 3)^a
10 weeks	763 ± 168
30 weeks	1500 ± 40
50 weeks	1270 ± 72
70 weeks	1500 ± 43

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^aAdduct levels in NNK-treated rats are from ref. (3).

Discussion

This is the first study to report the presence of methyl DNA phosphate adducts in humans. Using the optimized LC–NSI–HRMS/MS method, we were able to detect certain adducts as low as two adducts/10¹⁰ nucleobases in human lung DNA samples. We measured methyl DNA phosphate adducts in lung tumor and its adjacent tissue samples from 30 patients with lung cancer, including 13 current smokers and 17 current non-smokers at the time of sample collection. We compared the adduct levels between normal and tumor tissues from the same individuals, as well as the levels in normal lung tissues between smokers and non-smokers. Using the optimized method, we were also able to detect methyl DNA phosphate adducts in the lung tissues of untreated rats from our previous study, in which these adducts were not detected using the less sensitive method developed in that study (3).

In smokers, the levels of total methyl DNA phosphate adducts and some individual base combinations were higher in normal tissues than tumor tissues, whereas such difference was not observed in non-smokers. Higher adduct levels in normal tissue than tumor tissue were also observed for some other DNA adducts. A previous study examined the levels of bulky DNA adducts by ³²P-postlabelling and polycyclic aromatic hydrocarbon (PAH)-DNA adducts by immunoassay in tumor and normal lung tissues from patients with lung cancer. Both bulky DNA adducts and PAH-DNA adducts in smokers showed higher levels in normal lung tissues compared with tumor tissues (27). Adducts formed by 4-aminobiphenyl, a tobacco smoke constituent, were analyzed in patients with breast cancer, with higher adduct levels being observed in normal breast tissues than tumor tissues (28). The difference of adduct levels between normal and tumor tissues could result from their different metabolic rates of methylating agents, different DNA repair capacity, and/or ‘adduct dilution effect’ due to rapid proliferation in tumor tissues (27).

We observed 2-fold higher total adduct levels in the normal lung tissues of smokers compared with non-smokers, despite the contribution of adduct formation from endogenous and possibly environmental methylating agents. Our results suggest that smoking is a contributor to the formation of methyl DNA phosphate adducts in the lung of smokers, although further studies with larger numbers of subjects would be necessary to confirm this. There are several methylating agents present in tobacco products, including the tobacco-specific nitrosamine NNK, N-nitrosodimethylamine (NDMA) and methyl chloride (29), which can react with DNA forming methyl DNA adducts. Similar to our observation on DNA phosphate adducts, smoking also contributed to the formation of certain methyl base adducts with higher adduct levels observed in smokers compared with non-smokers. 7-Methylguanine was measured in human peripheral white blood cells and bronchial tissues, and the adduct levels in smokers were significantly higher than in non-smokers (30,31). Smoking also contributes to the formation of DNA adducts, which are formed by chemicals in tobacco smoke, including PAHs, aromatic and heterocyclic aromatic amines and aldehydes (1,32). However, the association of these adducts with tobacco smoke was often compromised by the presence of the same chemicals in the environment and by other confounding variables (32).

Compared with methyl DNA base adducts, methyl DNA phosphate adducts are generally more persistent in vivo with much longer half-lives, partially due to a lack of repair mechanism. Although methyl base adducts are repaired by O⁶-alkylguanine DNA alkyltransferase and base excision repair (13), only the (S) isomers of methyl DNA phosphate adducts are removed by O⁶-alkylguanine DNA alkyltransferase, leaving all the (R) isomers unrepaired in E.coli (33,34). However, the possible repair mechanisms in humans are still unknown. Those unrepaired isomers could contribute to the overall persistence of methyl DNA phosphate adducts in vivo and may serve as better biomarkers of chronic exposure to methylating agents. In this study, we did not identify which peaks are (R) or (S) isomers of methyl DNA phosphate adducts, and further studies should be conducted to identify these specific isomers. In our previous studies of rats treated with 5 ppm NNK or NNAL in their drinking water for 10, 30, 50 or 70 weeks, the levels of total methyl DNA phosphate adducts increased from 10 to 30 weeks and stayed constant afterward, whereas O⁶-methylguanine levels decreased over the course of the study (2,3). In a previous study of rats intraperitoneally injected with a single dose of NDMA, a half-life of 7 days in the liver was observed for the methyl DNA phosphate adduct TpMeT, compared with a half-life of 25 h for O⁶-methylguanine (35). Similar results were obtained in another study in mice treated with a methylating agent N-methyl-N-nitrosourea, in which the half-life of methyl DNA phosphate adducts was determined to be ~7 days in liver, lung and kidney, compared with O⁶-methylguanine with a half-life of 13 h (36). Such characteristic persistence was also observed in other types of DNA phosphate adducts such as ethyl (36), pyridyloxobutyl (37) and pyridylhydroxybutyl (38,39) DNA phosphate adducts. The constant levels of methyl DNA phosphate adducts in the untreated rats from this study and in NNK- or NNAL-treated rats from our previous study suggest that the adduct levels stay steady over time in the context of similar exposure levels. Taking into account the mutagenic properties and other biological effects, methyl DNA phosphate adducts could potentially serve as better biomarkers than DNA base adducts to represent chronic and consistent exposure to methylating agents. For example, humans are constantly exposed to very low levels of the methylating agent NDMA present in processed food and drinking water (40,41). The methyl DNA phosphate adducts possibly formed by NDMA could be biomarkers to investigate the potential biological consequences of chronic NDMA exposure.

In addition to environmental and endogenous methylating agents, medicinal methylating agents are another exposure source in patients who are given chemotherapeutic drugs such as dacarbazine and temozolomide (8,9). The therapeutic mechanism of those drugs is the formation of methyldiazonium ions, which react with DNA nucleobases to form methyl DNA base adducts. The cytotoxicity of the drugs is primarily mediated by O⁶-mdG, which generates a base mismatch eventually leading to cell death (8). However, the formation of methyl DNA phosphate adducts and their contribution to cytotoxicity have never been investigated. The formation of methyl DNA phosphate adducts by NNK and NNAL is mediated by methyldiazonium ions (3), which are also generated by dacarbazine and temozolomide. Therefore, methyl DNA phosphate adducts are probably also formed by these two drugs and further studies are warranted.

Human lung tissues were used for the analysis of methyl DNA phosphate adducts in this study. However, obtaining human lung tissue especially from ‘healthy’ individuals for exposure evaluation and DNA adduct analysis is highly impractical in larger studies. Surrogate tissues, such as bronchoalveolar lavage, peripheral white blood cells or oral cells, can be less invasively obtained and could be explored in future studies. For example, bronchoalveolar lavage samples have been used to investigate the formation of PAH-DNA adducts in humans (42). Oral cell DNA is another source of readily obtained biological samples for DNA adduct analysis and studies have demonstrated a correlation between changes in the oral cavity and lung in smokers (43,44).

In summary, for the first time, we detected methyl DNA phosphate adducts in human lung using our optimized LC–NSI–HRMS/MS method. Methyl DNA phosphate adducts were quantified in lung tumor tissue and its adjacent tissue from 13 smokers and 17 non-smokers, as well as lung tissues from untreated rats. Adduct levels in normal lung tissues were higher in smokers than non-smokers, suggesting that smoking is one contributor to adduct formation. Levels of methyl DNA phosphate adducts in rat lung tissues were consistent over the course of the study, indicating the persistence of these adducts. The detection and quantitation of methyl DNA phosphate adducts in human lung tissues provide the first insight of these adducts as biomarkers to investigate methylating agent exposure-associated carcinogenesis in humans.

Funding

National Cancer Institute [CA-81301]. Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, supported in part by Cancer Center Support Grant from National Cancer Institute [CA-77598]. Salary support for P.W.V. was provided by National Cancer Institute [CA-211256].

Conflict of interest: None declared.

Supplementary Material

bgz053_suppl_Supplementary_Material

Click here for additional data file.^{(91.9KB, docx)}

Acknowledgements

We thank Leah Raddatz and Dr Sharon Murphy for their help with urine sample analysis for total cotinine and total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. We thank Dr Naomi Fujioka for helpful discussions on the results and future directions. We also thank Bob Carlson for editorial assistance.

Glossary

Abbreviations

7-mdG: 7-methyl-2′-deoxyguanosine
LC–NSI–HRMS/MS: liquid chromatography–nanoelectrospray ionization–high-resolution tandem mass spectrometry
NNK: 4-(methylnitrosamino)-1- (3-pyridyl)-1- butanone
NNAL: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
NDMA: N-nitrosodimethylamine
PAH: polycyclic aromatic hydrocarbon
TpMeT: thymidylyl(3′-5′)thymidine methyl phosphotriester

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9. Zhang J., et al. (2012) Temozolomide: mechanisms of action, repair and resistance. Curr. Mol. Pharmacol., 5, 102–114. [DOI] [PubMed] [Google Scholar]
10. Peterson L.A. (2017) Context matters: contribution of specific DNA adducts to the genotoxic properties of the tobacco-specific nitrosamine NNK. Chem. Res. Toxicol., 30, 420–433. [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Earley L.F., et al. (2013) Mutagenic spectra arising from replication bypass of the 2,6-diamino-4-hydroxy-N(5)-methyl formamidopyrimidine adduct in primate cells. Chem. Res. Toxicol., 26, 1108–1114. [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Fu D., et al. (2012) Balancing repair and tolerance of DNA damage caused by alkylating agents. Nat. Rev. Cancer, 12, 104–120. [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Wyatt M.D., et al. (2006) Methylating agents and DNA repair responses: methylated bases and sources of strand breaks. Chem. Res. Toxicol., 19, 1580–1594. [DOI] [PMC free article] [PubMed] [Google Scholar]
14. Shrivastav N., et al. (2010) Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation. Carcinogenesis, 31, 59–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Pauly G.T., et al. (2001) Mutagenesis by O(6)-methyl-, O(6)-ethyl-, and O(6)-benzylguanine and O(4)-methylthymine in human cells: effects of O(6)-alkylguanine-DNA alkyltransferase and mismatch repair. Chem. Res. Toxicol., 14, 894–900. [DOI] [PubMed] [Google Scholar]
16. Jones G.D., et al. (2010) Phosphotriester adducts (PTEs): DNA’s overlooked lesion. Mutagenesis, 25, 3–16. [DOI] [PubMed] [Google Scholar]
17. Suhasini A.N., et al. (2012) DNA repair and replication fork helicases are differentially affected by alkyl phosphotriester lesion. J. Biol. Chem., 287, 19188–19198. [DOI] [PMC free article] [PubMed] [Google Scholar]
18. He C., et al. (2005) A methylation-dependent electrostatic switch controls DNA repair and transcriptional activation by E. coli ada. Mol. Cell, 20, 117–129. [DOI] [PubMed] [Google Scholar]
19. Wu J., et al. (2018) Cytotoxic and mutagenic properties of alkyl phosphotriester lesions in Escherichia coli cells. Nucleic Acids Res., 46, 4013–4021. [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Hecht S.S., et al. (2016) Exposure and metabolic activation biomarkers of carcinogenic tobacco-specific nitrosamines. Acc. Chem. Res., 49, 106–114. [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Carmella S.G., et al. (2013) High throughput liquid and gas chromatography-tandem mass spectrometry assays for tobacco-specific nitrosamine and polycyclic aromatic hydrocarbon metabolites associated with lung cancer in smokers. Chem. Res. Toxicol., 26, 1209–1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
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23. Lao Y., et al. (2007) Formation and accumulation of pyridyloxobutyl DNA adducts in F344 rats chronically treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and enantiomers of its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Chem. Res. Toxicol., 20, 235–245. [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Villalta P.W., et al. (2017) Ultrasensitive high-resolution mass spectrometric analysis of a DNA adduct of the carcinogen benzo[a]pyrene in human lung. Anal. Chem., 89, 12735–12742. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Bansal M. (2003) DNA structure: revisiting the Watson-Crick double helix. Curr. Sci., 85, 1556–1563. [Google Scholar]
26. Klaene J.J., et al. (2016) Tracking matrix effects in the analysis of DNA adducts of polycyclic aromatic hydrocarbons. J. Chromatogr. A, 1439, 112–123. [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Gyorffy E., et al. (2004) DNA adducts in tumour, normal peripheral lung and bronchus, and peripheral blood lymphocytes from smoking and non-smoking lung cancer patients: correlations between tissues and detection by 32P-postlabelling and immunoassay. Carcinogenesis, 25, 1201–1209. [DOI] [PubMed] [Google Scholar]
28. Faraglia B., et al. (2003) Evaluation of 4-aminobiphenyl-DNA adducts in human breast cancer: the influence of tobacco smoke. Carcinogenesis, 24, 719–725. [DOI] [PubMed] [Google Scholar]
29. Scherer G., et al. (2010) Determination of methyl-, 2-hydroxyethyl- and 2-cyanoethylmercapturic acids as biomarkers of exposure to alkylating agents in cigarette smoke. J. Chromatogr. B, 878, 2520–2528. [DOI] [PubMed] [Google Scholar]
30. Mustonen R., et al. (1992) 7-Methylguanine levels in DNA of smokers’ and non-smokers’ total white blood cells, granulocytes and lymphocytes. Carcinogenesis, 13, 1951–1955. [DOI] [PubMed] [Google Scholar]
31. Mustonen R., et al. (1993) Smoking-related DNA adducts: 32P-postlabeling analysis of 7-methylguanine in human bronchial and lymphocyte DNA. Carcinogenesis, 14, 151–154. [DOI] [PubMed] [Google Scholar]
32. Phillips D.H., et al. (2012) DNA and protein adducts in human tissues resulting from exposure to tobacco smoke. Int. J. Cancer, 131, 2733–2753. [DOI] [PubMed] [Google Scholar]
33. Weinfeld M., et al. (1985) Stereospecific removal of methyl phosphotriesters from DNA by an Escherichia coli ada+ extract. Nucleic Acids Res., 13, 7067–7077. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. McCarthy T.V., et al. (1985) Methyl phosphotriesters in alkylated DNA are repaired by the Ada regulatory protein of E. coli. Nucleic Acids Res., 13, 2683–2698. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Den Engelse L., et al. (1986) Formation and stability of alkylated pyrimidines and purines (including imidazole ring-opened 7-alkylguanine) and alkylphosphotriesters in liver DNA of adult rats treated with ethylnitrosourea or dimethylnitrosamine. Carcinogenesis, 7, 393–403. [DOI] [PubMed] [Google Scholar]
36. Shooter K.V., et al. (1977) The stability of methyl and ethyl phosphotriesters in DNA in vivo. Chem. Biol. Interact., 19, 353–361. [DOI] [PubMed] [Google Scholar]
37. Ma B., et al. (2015) Comprehensive high-resolution mass spectrometric analysis of DNA phosphate adducts formed by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Chem. Res. Toxicol., 28, 2151–2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Ma B., et al. (2018) Identification of more than 100 structurally unique DNA-phosphate adducts formed during rat lung carcinogenesis by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Carcinogenesis, 39, 232–241. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Ma B., et al. (2017) Pyridylhydroxybutyl and pyridyloxobutyl DNA phosphate adduct formation in rats treated chronically with enantiomers of the tobacco-specific nitrosamine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Mutagenesis, 32, 561–570. [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Krasner S.W., et al. (2013) Formation, precursors, control, and occurrence of nitrosamines in drinking water: a review. Water Res., 47, 4433–4450. [DOI] [PubMed] [Google Scholar]
41. Dybing E., et al. (2008) Risk assessment of dietary exposures to compounds that are genotoxic and carcinogenic–an overview. Toxicol. Lett., 180, 110–117. [DOI] [PubMed] [Google Scholar]
42. Piipari R., et al. (2000) Expression of CYP1A1, CYP1B1 and CYP3A, and polycyclic aromatic hydrocarbon-DNA adduct formation in bronchoalveolar macrophages of smokers and non-smokers. Int. J. Cancer, 86, 610–616. [DOI] [PubMed] [Google Scholar]
43. Hecht S.S. (2017) Oral cell DNA adducts as potential biomarkers for lung cancer susceptibility in cigarette smokers. Chem. Res. Toxicol., 30, 367–375. [DOI] [PMC free article] [PubMed] [Google Scholar]
44. Bhutani M., et al. (2008) Oral epithelium as a surrogate tissue for assessing smoking-induced molecular alterations in the lungs. Cancer Prev. Res. (Phila)., 1, 39–44. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[CIT0009] 9. Zhang J., et al. (2012) Temozolomide: mechanisms of action, repair and resistance. Curr. Mol. Pharmacol., 5, 102–114. [DOI] [PubMed] [Google Scholar]

[CIT0010] 10. Peterson L.A. (2017) Context matters: contribution of specific DNA adducts to the genotoxic properties of the tobacco-specific nitrosamine NNK. Chem. Res. Toxicol., 30, 420–433. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0011] 11. Earley L.F., et al. (2013) Mutagenic spectra arising from replication bypass of the 2,6-diamino-4-hydroxy-N(5)-methyl formamidopyrimidine adduct in primate cells. Chem. Res. Toxicol., 26, 1108–1114. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0012] 12. Fu D., et al. (2012) Balancing repair and tolerance of DNA damage caused by alkylating agents. Nat. Rev. Cancer, 12, 104–120. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0013] 13. Wyatt M.D., et al. (2006) Methylating agents and DNA repair responses: methylated bases and sources of strand breaks. Chem. Res. Toxicol., 19, 1580–1594. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0014] 14. Shrivastav N., et al. (2010) Chemical biology of mutagenesis and DNA repair: cellular responses to DNA alkylation. Carcinogenesis, 31, 59–70. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0015] 15. Pauly G.T., et al. (2001) Mutagenesis by O(6)-methyl-, O(6)-ethyl-, and O(6)-benzylguanine and O(4)-methylthymine in human cells: effects of O(6)-alkylguanine-DNA alkyltransferase and mismatch repair. Chem. Res. Toxicol., 14, 894–900. [DOI] [PubMed] [Google Scholar]

[CIT0016] 16. Jones G.D., et al. (2010) Phosphotriester adducts (PTEs): DNA’s overlooked lesion. Mutagenesis, 25, 3–16. [DOI] [PubMed] [Google Scholar]

[CIT0017] 17. Suhasini A.N., et al. (2012) DNA repair and replication fork helicases are differentially affected by alkyl phosphotriester lesion. J. Biol. Chem., 287, 19188–19198. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0018] 18. He C., et al. (2005) A methylation-dependent electrostatic switch controls DNA repair and transcriptional activation by E. coli ada. Mol. Cell, 20, 117–129. [DOI] [PubMed] [Google Scholar]

[CIT0019] 19. Wu J., et al. (2018) Cytotoxic and mutagenic properties of alkyl phosphotriester lesions in Escherichia coli cells. Nucleic Acids Res., 46, 4013–4021. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0020] 20. Hecht S.S., et al. (2016) Exposure and metabolic activation biomarkers of carcinogenic tobacco-specific nitrosamines. Acc. Chem. Res., 49, 106–114. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0021] 21. Carmella S.G., et al. (2013) High throughput liquid and gas chromatography-tandem mass spectrometry assays for tobacco-specific nitrosamine and polycyclic aromatic hydrocarbon metabolites associated with lung cancer in smokers. Chem. Res. Toxicol., 26, 1209–1217. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0022] 22. Murphy S.E., et al. (2014) Nicotine N-glucuronidation relative to N-oxidation and C-oxidation and UGT2B10 genotype in five ethnic/racial groups. Carcinogenesis, 35, 2526–2533. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0023] 23. Lao Y., et al. (2007) Formation and accumulation of pyridyloxobutyl DNA adducts in F344 rats chronically treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and enantiomers of its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Chem. Res. Toxicol., 20, 235–245. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0024] 24. Villalta P.W., et al. (2017) Ultrasensitive high-resolution mass spectrometric analysis of a DNA adduct of the carcinogen benzo[a]pyrene in human lung. Anal. Chem., 89, 12735–12742. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0025] 25. Bansal M. (2003) DNA structure: revisiting the Watson-Crick double helix. Curr. Sci., 85, 1556–1563. [Google Scholar]

[CIT0026] 26. Klaene J.J., et al. (2016) Tracking matrix effects in the analysis of DNA adducts of polycyclic aromatic hydrocarbons. J. Chromatogr. A, 1439, 112–123. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0027] 27. Gyorffy E., et al. (2004) DNA adducts in tumour, normal peripheral lung and bronchus, and peripheral blood lymphocytes from smoking and non-smoking lung cancer patients: correlations between tissues and detection by 32P-postlabelling and immunoassay. Carcinogenesis, 25, 1201–1209. [DOI] [PubMed] [Google Scholar]

[CIT0028] 28. Faraglia B., et al. (2003) Evaluation of 4-aminobiphenyl-DNA adducts in human breast cancer: the influence of tobacco smoke. Carcinogenesis, 24, 719–725. [DOI] [PubMed] [Google Scholar]

[CIT0029] 29. Scherer G., et al. (2010) Determination of methyl-, 2-hydroxyethyl- and 2-cyanoethylmercapturic acids as biomarkers of exposure to alkylating agents in cigarette smoke. J. Chromatogr. B, 878, 2520–2528. [DOI] [PubMed] [Google Scholar]

[CIT0030] 30. Mustonen R., et al. (1992) 7-Methylguanine levels in DNA of smokers’ and non-smokers’ total white blood cells, granulocytes and lymphocytes. Carcinogenesis, 13, 1951–1955. [DOI] [PubMed] [Google Scholar]

[CIT0031] 31. Mustonen R., et al. (1993) Smoking-related DNA adducts: 32P-postlabeling analysis of 7-methylguanine in human bronchial and lymphocyte DNA. Carcinogenesis, 14, 151–154. [DOI] [PubMed] [Google Scholar]

[CIT0032] 32. Phillips D.H., et al. (2012) DNA and protein adducts in human tissues resulting from exposure to tobacco smoke. Int. J. Cancer, 131, 2733–2753. [DOI] [PubMed] [Google Scholar]

[CIT0033] 33. Weinfeld M., et al. (1985) Stereospecific removal of methyl phosphotriesters from DNA by an Escherichia coli ada+ extract. Nucleic Acids Res., 13, 7067–7077. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0034] 34. McCarthy T.V., et al. (1985) Methyl phosphotriesters in alkylated DNA are repaired by the Ada regulatory protein of E. coli. Nucleic Acids Res., 13, 2683–2698. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0035] 35. Den Engelse L., et al. (1986) Formation and stability of alkylated pyrimidines and purines (including imidazole ring-opened 7-alkylguanine) and alkylphosphotriesters in liver DNA of adult rats treated with ethylnitrosourea or dimethylnitrosamine. Carcinogenesis, 7, 393–403. [DOI] [PubMed] [Google Scholar]

[CIT0036] 36. Shooter K.V., et al. (1977) The stability of methyl and ethyl phosphotriesters in DNA in vivo. Chem. Biol. Interact., 19, 353–361. [DOI] [PubMed] [Google Scholar]

[CIT0037] 37. Ma B., et al. (2015) Comprehensive high-resolution mass spectrometric analysis of DNA phosphate adducts formed by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Chem. Res. Toxicol., 28, 2151–2159. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0038] 38. Ma B., et al. (2018) Identification of more than 100 structurally unique DNA-phosphate adducts formed during rat lung carcinogenesis by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Carcinogenesis, 39, 232–241. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0039] 39. Ma B., et al. (2017) Pyridylhydroxybutyl and pyridyloxobutyl DNA phosphate adduct formation in rats treated chronically with enantiomers of the tobacco-specific nitrosamine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Mutagenesis, 32, 561–570. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0040] 40. Krasner S.W., et al. (2013) Formation, precursors, control, and occurrence of nitrosamines in drinking water: a review. Water Res., 47, 4433–4450. [DOI] [PubMed] [Google Scholar]

[CIT0041] 41. Dybing E., et al. (2008) Risk assessment of dietary exposures to compounds that are genotoxic and carcinogenic–an overview. Toxicol. Lett., 180, 110–117. [DOI] [PubMed] [Google Scholar]

[CIT0042] 42. Piipari R., et al. (2000) Expression of CYP1A1, CYP1B1 and CYP3A, and polycyclic aromatic hydrocarbon-DNA adduct formation in bronchoalveolar macrophages of smokers and non-smokers. Int. J. Cancer, 86, 610–616. [DOI] [PubMed] [Google Scholar]

[CIT0043] 43. Hecht S.S. (2017) Oral cell DNA adducts as potential biomarkers for lung cancer susceptibility in cigarette smokers. Chem. Res. Toxicol., 30, 367–375. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CIT0044] 44. Bhutani M., et al. (2008) Oral epithelium as a surrogate tissue for assessing smoking-induced molecular alterations in the lungs. Cancer Prev. Res. (Phila)., 1, 39–44. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Methyl DNA phosphate adduct formation in lung tumor tissue and adjacent normal tissue of lung cancer patients

Bin Ma

Peter W Villalta

J Bradley Hochalter

Irina Stepanov

Stephen S Hecht

Abstract

Introduction