|Comparative analysis of four oxidized guanine lesions from reactions of DNA with peroxynitrite, singlet oxygen, and γ-radiation.|
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|PMID: 23140136 Owner: NLM Status: MEDLINE|
|Oxidative damage to DNA has many origins, including irradiation, inflammation, and oxidative stress, but the chemistries are not the same. The most oxidizable base in DNA is 2-deoxyguanosine (dG), and the primary oxidation products are 8-oxodG and 2-amino-imidazolone. The latter rapidly converts to 2,2-diamino-oxazolone (Ox), and 8-oxodG is further oxidized to spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). In this study, we have examined the dose-response relationship for the formation of the above four products arising in calf thymus DNA exposed to gamma irradiation, photoactivated rose bengal, and two sources of peroxynitrite. In order to carry out these experiments, we developed a chromatographic system and synthesized isotopomeric internal standards to enable accurate and precise analysis based upon selected reaction monitoring mass spectrometry. 8-OxodG was the most abundant products in all cases, but its accumulation was highly dependent on the nature of the oxidizing agent and the subsequent conversion to Sp and Gh. Among the other oxidation products, Ox was the most abundant, and Sp was formed in significantly greater yield than Gh.|
|Liang Cui; Wenjie Ye; Erin G Prestwich; John S Wishnok; Koli Taghizadeh; Peter C Dedon; Steven R Tannenbaum|
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|Type: Comparative Study; Journal Article; Research Support, N.I.H., Extramural Date: 2013-01-24|
|Title: Chemical research in toxicology Volume: 26 ISSN: 1520-5010 ISO Abbreviation: Chem. Res. Toxicol. Publication Date: 2013 Feb|
|Created Date: 2013-02-18 Completed Date: 2013-07-29 Revised Date: 2014-03-24|
Medline Journal Info:
|Nlm Unique ID: 8807448 Medline TA: Chem Res Toxicol Country: United States|
|Languages: eng Pagination: 195-202 Citation Subset: IM|
|APA/MLA Format Download EndNote Download BibTex|
DNA / chemistry*
Deoxyguanosine / analogs & derivatives, chemistry
Guanidines / chemistry
Guanine / chemistry*
Guanosine / analogs & derivatives, chemistry
Hydantoins / chemistry
Oxidants / chemistry*, toxicity
Peroxynitrous Acid / chemistry*, toxicity
Rose Bengal / chemistry, toxicity
Singlet Oxygen / chemistry*, toxicity
Spiro Compounds / chemistry
|5T32-ES007020-34/ES/NIEHS NIH HHS; CA26731/CA/NCI NIH HHS; ES002109/ES/NIEHS NIH HHS; P01 CA026731/CA/NCI NIH HHS; T32 ES007020/ES/NIEHS NIH HHS|
|0/Guanidines; 0/Hydantoins; 0/Oxidants; 0/Spiro Compounds; 0/guanidinohydantoin; 0/spiroiminodihydantoin; 12133JR80S/Guanosine; 14691-52-2/Peroxynitrous Acid; 17778-80-2/Singlet Oxygen; 1ZPG1ELY14/Rose Bengal; 5Z93L87A1R/Guanine; 88847-89-6/8-oxo-7-hydrodeoxyguanosine; 9007-49-2/DNA; 91080-16-9/calf thymus DNA; G9481N71RO/Deoxyguanosine|
Journal ID (nlm-ta): Chem Res Toxicol
Journal ID (iso-abbrev): Chem. Res. Toxicol
Journal ID (publisher-id): tx
Journal ID (coden): crtoec
Publisher: American Chemical Society
Copyright © 2012 American Chemical Society
Received Day: 30 Month: 06 Year: 2012
Electronic publication date: Day: 09 Month: 11 Year: 2012
Print publication date: Day: 18 Month: 02 Year: 2013
Volume: 26 Issue: 2
First Page: 195 Last Page: 202
PubMed Id: 23140136
|Comparative Analysis of Four Oxidized Guanine Lesions from Reactions of DNA with Peroxynitrite, Singlet Oxygen, and γ-Radiation|
|Erin G. Prestwich†|
|John S. Wishnok†‡|
|Peter C. Dedon†‡|
|Steven R. Tannenbaum*†‡§|
of Biological Engineering, ‡Center for Environmental Health Sciences, and §Department of
Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United
and Technology, Center for Life Sciences, National University
of Singapore, 28 Medical Drive, Singapore 117456
of Biological Engineering,
56-731A, Massachusetts Institute of Technology, 77 Massachusetts Avenue,
Cambridge, MA 02139. Tel: 617-253-3729. E-mail: email@example.com.
DNA oxidation plays a significant role in the pathophysiology of cancers, aging, and inherited diseases,1−3 with epidemiological studies demonstrating a strong association between the reactive oxygen (ROS) and nitrogen species (RNS) of chronic inflammation and increased cancer risk.1−6 This potential for DNA damage to play a causative role in mutation and carcinogenesis has prompted the chemical characterization of oxidatively induced DNA damage and the quest to develop biomarkers that reflect this damage and serve as surrogates for short-lived ROS and RNS.7 As the most readily oxidized site in DNA, guanine is a major target for DNA oxidants, and its oxidation produces a wide spectrum of end products,8 including 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) spiroiminodihydantoin (Sp) guanidinohydantoin (Gh), 2-amino-5-[2-deoxy-β-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (imidazolone) and its hydrolysis product, 2,2-diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (Ox) (Scheme 1(9)). Although 8-oxodG is produced abundantly in DNA exposed to RNS and ROS,10−16 it is several orders-of-magnitude more susceptible to further oxidation than dG itself,17 yielding more stable secondary oxidation products, including Sp and Gh (Scheme 1).13,18,19 Ox and its precursor imidazolone have been observed as oxidation products of dG or 8-oxodG in oligodeoxynucleotides and calf thymus DNA treated with Mn-TMPyP/KHSO5,20 peroxynitrite (ONOO–),19,21 and photochemical oxidants22−25 (Scheme 1). All of these DNA oxidation products are promutagenic,10,26−30 with both diastereoisomers of Sp strongly inhibiting DNA polymerase27,31 and causing mutation frequencies that are at least an order of magnitude higher than those for 8-oxodG.
Therefore, as part of our current focus on inflammation-related DNA damage, including assessment of potential biomarkers, we developed a liquid chromatography-coupled isotope-dilution triple-quadrupole mass spectrometry method (LC-MS/MS) to simultaneously measure 8-oxodG, Sp, Gh, and Ox in DNA and applied the method to DNA treated in vitro with ONOO–, singlet oxygen derived from photoactivated rose bengal, and γ-radiation.
Singlet oxygen, with a high quantum yield in aqueous solution, is an important reactive oxygen species in vivo and, although accompanied by low levels of other reactive species, is the primary oxidant arising via rose bengal photosensitization.32,33 γ-Irradiation of aqueous solutions, however, yields hydroxyl radicals and hydrated electrons, with the former causing oxidation of both 2′-deoxyribose and nucleobases in DNA.34,35 ONOO– is the product of a reaction of macrophage-derived nitric oxide and superoxide, and its protonated form undergoes homolytic bond scission to yield a hydroxyl radical and a nitrogen dioxide radical, which are strong and weak oxidants, respectively.36 The new LC-MS/MS method, which is both more efficient and more sensitive than previous methods, also revealed novel features of the oxidant-specific distribution of these G oxidation products.
Uniformly labeled dG (13C and 15N > 98%) and uniformly labeled 15N dG (≥98%) were obtained from Cambridge Isotope Laboratories (Andover, MA). Benzonase nuclease, deoxyribonuclease (DNase), calf intestine alkaline phosphatase, and calf thymus DNA were from Sigma Aldrich (St. Louis, MO), ONOO– solution from Cayman Chemical Company (Ann Arbor, MI), phosphodiesterase 1 from USB Products (Cleveland, OH), Nanosep Omega 10 kD exclusion filter columns from Pall Corporation (Ann Arbor, MI), and Sep-Pak C18 cartridges from Waters (Milford, Massachusetts). All other chemicals, reagents, and solvents were purchased from Sigma-Aldrich. UV/vis spectroscopy measurements were made on an HP8452 diode-array spectrophotometer (Agilent Technologies, Palo Alto, CA) or a Beckman DU640 diode-array spectrophotometer (Beckman Coulter, Inc., Indianapolis, IN). High-performance liquid chromatography (HPLC) analyses were carried out on an Agilent 1100 Series HPLC system with binary pumps, a degasser, and an autoinjector. Quantitative liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses were conducted on an Agilent 6430 triple quadrupole mass spectrometer interfaced with an Agilent capillary 1200 binary pumping system or an AB Sciex API 3000 triple quadrupole mass spectrometer (AB Sciex, Framingham, MA) interfaced with an Agilent 1100 binary HPLC. Electrospray ionization mass spectrometry analyses for isotopic purities and structure confirmations by exact mass were done on an Agilent MSD-TOF. Experiments were designed to yield similar levels of Sp across the different oxidation conditions in order to facilitate comparisons. This approach provides a means for comparing relative amounts of the other lesions rather than total amounts of damage.
15N-Labeled 8-oxodG was synthesized as described earlier,37,38 and its identity was confirmed by comparison of its reversed phase HPLC retention time with that of commercially available 8-oxodG, its characteristic UV spectrum (λmax= 248 and 292 nm), and by ESI-MS/MS, which gave a protonated molecular ion with m/z 289 and two fragment ions at m/z 117 and m/z 173, which correspond to the protonated 2′-deoxyribose and 15N5-8-oxo-guanine. The standard was quantified by UV spectroscopy using an extinction coefficient of 10,300 M–1 cm–1 at 293 nm.39
The 13C-, 15N-labeled, and unlabeled Sp, Gh, and Ox were synthesized from labeled dG as described previously,21 and structures were confirmed by UV spectra, HPLC retention times, and ESI-MS/MS. For Sp, ESI-MS/MS analysis yielded a protonated molecular ion at m/z 300.1, along with characteristic fragment ions including m/z 184 and m/z 117.1 (Figure S1b). The molar extinction coefficient for the Sp was determined to be 10,500 M–1 cm–1 at λmax = 230 nm, which is consistent with the reported value.21 For Gh, the ESI-MS/MS spectrum (Figure S1c) showed m/z 158.0 as the major fragment released from the protonated Gh (m/z 274.1). For Ox, ESI-MS/MS analysis revealed a protonated molecular ion at m/z 246.7, with fragmentation occurring by both loss of 2-deoxyribose (m/z 131) and loss of CO2 ([M + H – CO2]+; m/z 202.9) (Figure S1d). This distinct fragmentation pattern reflects the loss of aromaticity upon oxidation of dG to oxazolone, accompanied by the incorporation of a relatively labile lactone group in the molecule.9 The major fragment at m/z 87.0 predominated [BH + H – CO2]+ (Figure S1d).
Working solutions of ONOO– were prepared in 0.1 M NaOH by dilution of the commercial stock solution of 100 mM in 0.3 M NaOH, the concentration of which was verified by measuring the absorbance at 302 nm (ε = 1670 M–1 cm–1).40 Aliquots of the working stock solutions of ONOO– were placed on the side walls of tubes containing a solution of calf thymus DNA (300 μg in 0.4 mL of 25 mM sodium phosphate buffer, pH 7.2) to produce final concentrations of 0–1000 μM. The oxidation reaction was initiated by rapidly vortexing the tube for 1 min and then allowing the reaction to proceed at ambient temperature for 1 h.
Calf thymus DNA (300 μg, 1 μg/μL in 50 mM sodium phosphate buffer, pH 7.4) was irradiated in the presence or absence of 10 μM rose bengal (added from a dimethyl sulfoxide (DMSO) stock solution) with UV light in a Rayonet RMR-600 reactor with RPR-3500 bulbs (λ max = 350 nm) for 0, 15, 30, 60, or 90 min. The control samples were treated with an equal volume of DMSO and irradiated with UV light for 90 min.
Calf thymus DNA (250 μg, 1 μg/μL) in Chelex-treated 50 mM sodium phosphate buffer at pH 7.4 was exposed to 60Co γ-radiation delivered at 133 Gy/min in a Gammacel-220 (MDS Nordion, Ottawa, Ontario, Canada), at ambient temperature, for total doses of 0, 7.5, 20, 33, 70, or 133 Gy.
Following each of the above treatments, DNA was precipitated by the addition of ethanol to 70%, washed with 70% ethanol, resuspended in 100 μL of water, and quantitated by UV/vis spectroscopy. The DNA was then digested to nucleosides with 4 units of benzonase nuclease, 0.1 units phosphodiesterase 1, 4 units DNase, and 20 units calf intestine alkaline phosphatase in 10 mM Tris buffer (pH 7.9) and 1 mM magnesium chloride. Deferoxamine mesylate (3 mM) and butylated hydroxytoluene (1 mM) were added as antioxidants to inhibit artifactual oxidation. 15N5-8-oxodG (1 pmol), 13C815N4-oxazolone (4.25 pmol), 13C915N5-guanidinohydantoin (5 pmol), and 13C1015N5-spiroiminodihydantoin (2.9 pmol) were added as internal standards and the samples digested for 4 h at 37 °C. Samples were then filtered through 10 kD exclusion filters to remove enzymes and, finally, dried under reduced pressure.
Digested samples were prepurified using a Varian Microsorb-MV C18 reversed-phase column (5 μm particle, 100 Å pore size, 250 × 4.6 mm) with an acetonitrile/8 mM ammonium acetate gradient. The prepurifications were performed with single 100 μL injections of hydrolyzed DNA samples. Individual fractions were then dried and subjected to LC-MS/MS analysis. The column was maintained at 10 °C with 0.6 mL/min flow rate, and the gradient conditions are detailed in Supporting Information, Table S1. Sp, Gh, and Ox were collected in a single fraction eluting between 3 and 6 min, and 8-oxodG was collected at 14.6–15.6 min (Supporting Information, Table S2). The fractions were dried under reduced pressure.
To control for DNA losses sustained during DNA workup, canonical 2′-deoxyribonucleosides were quantified by integrating the UV absorbance peaks obtained during HPLC fractionation and summing areas to compare to the 2′-deoxyribonucleoside calibration curves run with each experiment.
The dried samples containing 8-oxodG and the corresponding isotopomer were dissolved in 100 μL of deionized water, and 10 μL were analyzed on an Agilent 6410 tandem quadrupole, or an AB Sciex API 3000, interfaced with an Agilent 1100 series HPLC system. Residual dG was separated from 8-oxodG using isocratic reversed phase chromatography with 200 μL/min, 25 °C, 3% acetonitrile in water with 0.1% acetic acid on a C18 Thermo hypersil gold AQ column (150 × 2, 1 mm, 3 μm particles). It is important to note that 8-oxodG can be formed from dG in-source during ionization; therefore, separation of dG from 8-oxodG is essential for accurate quantitation.41,42 The mass spectrometers were operated in the positive ion mode, with all instrument parameters optimized for maximum sensitivity. Samples were analyzed in the multiple reaction monitoring mode (MRM), which monitors collision-induced dissociation of a precursor ion to an abundant characteristic product ion. The optimized parameters for the API 3000 were ion source, TurboIonSpray; 200 ms dwell time; declustering potential, 15 V; focusing potential, 90 V; entrance potential, 5 V; collision cell exit potential, 15 V; nebulizer gas, 13 AU (arbitrary unit); curtain gas, 7 AU; collision gas,: 9 AU; temperature, 400 °C; and ion spray voltage, 4,000 V. Samples were analyzed with the following transitions: m/z 284 → 168 for 8-oxodG, m/z 289 → m/z 173 for 15N5-labeled 8-oxodG, and m/z 268 → m/z 152 for dG.
The fraction containing Sp, Gh, Ox, and their corresponding isotopomers was dried and then redissolved in deionized water (30 μL). ESI-MS/MS analyses were done on an Agilent 6430 triple quadrupole mass spectrometer interfaced with an Agilent capillary 1200 series binary pumping system with a Hypercarb column (100 mm × 1.0 mm, 5 μm particles; Thermo Scientific, Torrance, CA). The gradient was 0.3% formic acid in water (A) and 0.3% formic acid in acetonitrile (B) from 2% to 70% B over 15 min at 20 μL/min (microionization spray capillary). The flow for the first 5 min was diverted to waste. The mass spectrometer was operated in positive ion mode, with instrument parameters optimized for maximal sensitivity (Supporting Information, Table S3). Samples were analyzed in multiple reaction monitoring mode (MRM), using the following transitions: m/z 300 → m/z 184 and m/z 315 → m/z 194 for Sp and uniformly 13C,15N-labeled Sp; m/z 274 → m/z 158 and m/z 288 → m/z 167 for Gh and uniformly 13C,15N-labeled Gh; m/z 247 → m/z 87 and m/z 259 → m/z 93 for Ox and uniformly 13C,15N-labeled Ox, respectively (Figure S1 and Supporting Information, Figure S4).
Calibration curves for the labeled and unlabeled forms of each of the four DNA oxidation products were constructed by plotting the MRM signal ratios between the labeled and unlabeled forms against their corresponding concentration ratios. Measurements of 8-oxodG, Sp, Gh, and Ox were based on the MRM signal ratios between analytes of interest and their isotope-labeled internal standards and their respective response curves (Table 1 and Supporting Information, Figure S5).
The limits of detection (LOD) and quantification (LOQ) of 8-oxodG, Ox, Sp, and Gh were determined by adding known amounts of the corresponding labeled nucleosides to deionized water, followed by LC-MS/MS analysis as described above. The nominal signal-to-noise ratios were ∼3 (LOD) and ∼10 (LOQ). Assuming approximately equal responses for the unlabeled and the labeled nucleosides in the MRM mode, the limit of detection of each oxidation product (the unlabeled nucleoside) was in the range of 1 to 3 fmol, with limits of quantification around 3 to 10 fmol, corresponding to approximately 1–3 nucleoside/108 nucleosides in 100 μg of DNA (Table 1)
The overall method workflow is outlined in Scheme 2. The first critical step in the method involves artifact-free processing of the oxidized DNA.7,21 In this case, treated DNA samples were purified by ethanol precipitation, followed by the addition of the antioxidants, deferoxamine mesylate and butylated hydroxytoluene, along with isotopically labeled internal standards. DNA was then enzymatically hydrolyzed with benzonase, phosphodiesterase 1, and deoxyribonuclease (DNase), with dephosphorylation to nucleosides by calf intestine alkaline phosphatase. Following the removal of enzymes by filtration, samples were subjected to HPLC purification of individual 2′-deoxyribonucleosides.
A second critical step in the method involves HPLC prepurification of the various damaged 2′-deoxyribonucleosides. The hydrophilic Sp, Gh, and Ox eluted early on the reversed-phase HPLC system and were collected as a single fraction, while 8-oxodG was well retained on the column. The two groups of 2′-deoxyribonucleosides were collected at the predetermined retention times noted in Experimental Procedures. To quantify the total DNA injected on the HPLC column, canonical 2′-deoxyribonucleosides were quantified by integrating the area under the peaks of the UV absorbance profiles obtained during the prepurification steps, with analysis by calibration curves.
The final step of the method involves LC-MS/MS analysis of the individual purified 2′-deoxyribonucleosides. The hydrophilic Sp, Gh, and Ox were optimally resolved on a Hypercarb graphite column using an acetonitrile/water gradient, while 8-oxodG was best resolved using a C18 reversed phase HPLC column, as in the prepurification. Both HPLC systems were coupled to a triple quadrupole mass spectrometer operated in positive ion mode. The Sp and Ox diastereomers were observed with this approach (Supporting Information, Figure S4). Typical detection limits for modified DNA nucleosides were 3–10 fmol (Table 1). For 8-oxodG analysis, the use of a reversed phase column eluted with acetic acid/acetonitrile provided similar MS/MS quantification properties as those reported previously.7
Several control studies were performed to assess oxidative artifacts during DNA processing and analysis. The detection and quantitation limits of the guanine oxidation products are listed in Table 1. The dG transition m/z 268 → m/z 152 was monitored to confirm the separation between 8-oxodG and dG and thus to ensure that artifactual production of 8-oxodG in the ion source is not included in the analysis.41,42 [13C10,14N5]-8-oxodG was not detected in the pilot experiments (1 nmol, in triplicate) spiked with [13C10,14N5]-dG, which suggests that artifactual formation of 8-oxodG during the sample processing was minimal. To assess the oxidation of 8-oxodG during workup, [15N5]-8-oxodG was added to a DNA sample before enzymatic digestion. Subsequent analysis for [15N5]-Sp, [15N5]-Gh, and [15N4]-Ox was below the detection limits, which demonstrates minimal artifactual formation of Sp, Gh, and Ox during the digestion and workup process.
The analytical method was now applied to quantify Sp, Gh, Ox, and 8-oxoG in DNA samples oxidized by four well studied oxidants: ONOO–, SIN-1, singlet oxygen arising from photoactivated rose bengal, and γ-radiation. The results are shown in Figures 1 and 2, Table 2, and in Supporting Information Tables S2 and S3. The background levels of damage products in the commercial calf thymus DNA were found to be 2.8 ± 1.4 8-oxodG per 105 nt, 4.7 ± 1.9 Sp per 108 nt, 0.52 ± 0.11 Gh per 108 nt, and 3.6 ± 6.2 Ox per 108 nt. The background of 8-oxodG in the calf thymus DNA is higher than the levels observed in cells and tissues43,44 but is consistent with literature values.45 As shown in Figure 1, SIN-1 produced nonlinear dose–response curves for all damage products, with ONOO–, γ-radiation, and rose bengal photosensitization producing more linear dose–response curves.
During chronic inflammation, locally produced ROS and RNS can attack DNA to cause mutation and cell death as part of the pathophysiology of cancer and other diseases.1−5 As the most readily oxidized of the nucleobases, guanine (G) is preferentially targeted by oxidants and produces a wide spectrum of end products, including 8-oxodG, Sp, Gh, and Ox. 8-OxodG is used widely as a biomarker of G oxidation, whereas other more stable oxidation products, such as Sp, Gh, and Ox, have received relatively little attention in spite of their toxicity and mutagenicity.46 A previously published method for LC-MS/MS analyses of 8-oxodG, Sp, Gh, and Ox21 was 10-times less sensitive for Sp and Gh than the present method, with an LOD of about ∼1 lesion per 107 nt. Further, the LC-MS/MS method of Sugden and co-workers, which used a reversed-phase HPLC resolution and no prepurification, yielded high background levels of Sp in wild-type E. coli cells on the order of 1 Sp per 104 to 105 nt.31 In the present studies, we have decreased the detection limits by at least 10-fold to the level of ∼1 lesion per 108 nt by employing a prepurification step and appropriate chromatographic systems for resolving the various damage products. In addition, our method gives similar results to those of earlier studies, e.g., for γ-radiation, confirming that our method is accurate.7,31,35,38,41−45 The more noteworthy result in the analysis of G oxidation products was the strong oxidant dependence of the product spectra. This is illustrated by the dose–response curves in Figure 1 and stacked distribution plots of the average G oxidation products under the different oxidizing conditions shown in Figure 2. Both figures were plotted to permit easy comparisons between oxidizing conditions and relative changes in G oxidation products. Clearly, 8-oxodG is the most abundant product formed with the four oxidants (Figure 1), with Ox consistently observed at higher levels than the other two secondary G oxidation products. These results are consistent with published reports,9,10,47 in which Ox was found to be the major secondary G oxidation product generated from one- electron oxidants, ONOO–, γ-radiation, and singlet oxygen.
Among individual oxidants, the lowest relative levels of 8-oxodG are present with photosensitized 1O2, which correlates with the high relative generation of Sp (Figure 1, Figure 2). Ye et al. reported that Sp was the major secondary oxidation product when guanosine was exposed to rose bengal-photosensitized 1O2 at neutral pH.48 Our results corroborate the prominence of Sp in 1O2-mediated chemistry, though more Ox was formed in our experiments than in the previous work. This difference is possibly due to the fact that Ye et al. performed their oxidation studies with 2′-deoxynucleosides, whereas we used calf thymus DNA. Previous studies have shown differences in relative ratios of different G oxidation products when using 2′-deoxyribonucleosides and single-stranded and double-stranded oligonucleotides.49 For γ-radiation, Figure 1A demonstrates linear increases in each G oxidation product examined, with Ox as the predominant product with relatively small proportions of Sp.
ONOO– appears to produce the most even distribution of G oxidation products under the conditions used, as shown in Figure 2. However, Ox remains the most abundant secondary G oxidation product (Table 2) and exists in higher relative proportion as compared to untreated DNA (Figure 2). The high rate of Ox production with ONOO– is substantiated by the reaction of DNA with SIN-1 that generates NO• and O2–• that combine to form ONOO–. The rate of ONOO– formation from SIN-1 decomposition varies with the medium.50 In phosphate buffer, the half-life of SIN-1 is estimated to be 14–26 min, with the release of ONOO– at a rate of 10–12 μM/min.51−53 Ox is formed in high relative rates (Figure 2) and rapidly increases with higher ONOO– concentrations. The concentrations of SIN-1 used in this experiment were relatively high compared to those in other studies examining DNA oxidation,54,55 which is likely the reason that ONOO– resulted in the highest overall levels of oxidation among the various oxidants (Table 2). Differences in the relative amounts of G oxidation products for ONOO– and SIN-1 are not surprising given the varying responses observed for the two oxidizing agents by Kim et al.,55 though that study was looking at more generalized damage. These differing responses are possibly due to other reactive and toxic products formed during the decomposition of SIN-156,57 in addition to the effects of sustained lower doses of ONOO– over time rather than bolus addition. Yu et al.21 observed a biphasic dose–response curve for 8-oxodG formation by ONOO– oxidation of DNA, with levels of 8-oxodG increasing up to 2 equivalents of ONOO–, then decreasing until 10 equivalents of ONOO– were added, and finally increasing again with higher concentrations of ONOO–. Further, they observed that Sp and Gh were formed in DNA in a dose-dependent manner at low ONOO– concentrations but that the levels of these lesions dropped significantly at high ONOO– concentrations. In contrast, the lower concentrations of ONOO– used in the present studies produced a nonlinear but smooth increase in the amounts of 8-oxo-dG, Sp, and Gh in DNA treated with increasing ONOO– concentrations. The differences could be partially due to the different dosage points monitored, increased number of experimental replicates performed, and higher ratio of dG to ONOO– in our studies. Yu et al. also observed more artifactual oxidation during digestion and workup as evidenced by the oxidation of labeled dG included in the analysis. When we performed a similar experiment, no oxidation of labeled dG was observed. The ONOO– used by Yu et al. synthesized via ozonolysis could have different impurities than that purchased for these studies.
In conclusion, a sensitive and specific LC-MS/MS method was developed for the quantification of 8-oxodG, Sp, Gh, and Ox lesions in DNA. Application of the method to DNA oxidized in vitro with four different oxidants revealed a clear oxidant dependence of the relative quantities of the G oxidation products.
Representative HPLC/UV chromatograms, LC-MS/MS chromatograms, calibration curves, HPLC gradients, elution profiles, LC-MS/MS parameters, standard synthesis schemes, and calculation methods. This material is available free of charge via the Internet at http://pubs.acs.org.
We acknowledge financial support from the National Cancer Institute (CA26731). Analytical studies were performed in the Bioanalytical Facilities Core of the MIT Center for Environmental Health Sciences, which is supported by a grant from the National Institute of Environmental Health Sciences (ES002109). E.G.P. was supported by an NIEHS Training Grant in Environmental Toxicology 5T32-ES007020-34.
The authors declare no competing financial interest.
We thank Agilent Technologies for graciously providing access to the 6430 triple quadrupole mass spectrometer system.
|LOD||limit of detection|
|LOQ||limit of quantification|
|ROS||reactive oxygen species|
|RNS||reactive nitrogen species|
|LC-MS/MS||liquid chromatography–tandem mass spectrometry|
|HPLC||high-performance liquid chromatography|
|MRM||reaction monitoring mode|
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[Figure ID: sch1]
Oxidation Products from dG by Various
[Figure ID: sch2]
Experimental Workflow for the Treatment
of DNA, Digestion, Sample
Preparation, and LC-MS/MS Analysis of 8-oxodG, Sp, Gh, and Ox
[Figure ID: fig2]
Dose–response curves for G oxidation products with different oxidants. DNA was treated with (a) γ-radiation, (b) ONOO–, (c) 1O2 from photooxidation of rose bengal, and (d) SIN-1, and the G oxidation products 8-oxodG, Sp, Gh, and Ox were quantified as described in Experimental Procedures. Data are plotted as the lesion level without exponential scaling on the Y-axis, with the exponential scaling noted in the inset. Data represent the mean ± SD.
[Figure ID: fig3]
Average amounts of each oxidation product across all measured doses for each oxidant were calculated and presented as a stack plot. The per nucleotide levels chosen were the same as those plotted in Figure 1.
|lesion||LOD (fmol)a||LOQ (fmol)a||minimum DNA (μg)||unlabeled standard (fmol)||internal standard (pmol)|
aLOD, limit of detection; LOQ, limit of quantitation.
|rose bengal irradiation time (min)||Sp per 108 nt||Gh per 108 nt||Ox per 108 nt||8-oxo per 105 nt|
|0||1.2 ± 0.1||0.7 ± 0.1||6.1 ± 1.7||2.5 ± 0.1|
|15||27 ± 1.7||4.2 ± 0.4||30 ± 3.7||4.5 ± 0.4|
|30||33 ± 1.3||5.7 ± 0.7||39 ± 4.5||4.5 ± 0.5|
|60||38 ± 7.1||4.8 ± 0.4||35 ± 7.3||6 ± 1.3|
|90||40 ± 3.6||7.6 ± 0.8||43 ± 5.8||5.7 ± 0.7|
|0||1.7 ± 0.2||0.7 ± 0.1||12 ± 0.08||1.4 ± 0.2|
|7.5||3.3 ± 0.2||1.0 ± 0.02||138 ± 2.6||3.9 ± 0.1|
|20||8.9 ± 0.9||1.3 ± 0.1||141 ± 11||6.3 ± 0.2|
|33||12 ± 1.2||1.9 ± 0.2||207 ± 6.2||8.3 ± 0.4|
|70||26 ± 1.0||3.7 ± 0.5||470 ± 10||18 ± 0.5|
|133||35 ± 0.3||7.4 ± 0.3||880 ± 12||27 ± 1.1|
|0||3.8 ± 0.7||0.7 ± 0.2||14 ± 0.9||4.4 ± 0.5|
|5||7.3 ± 1.4||1.3 ± 0.4||22 ± 4.9||6.1 ± 1.0|
|20||13 ± 0.4||1.8 ± 0.02||41 ± 1.5||9.8 ± 0.3|
|100||18 ± 0.9||3.7 ± 0.04||113 ± 13||18 ± 1.4|
|500||30 ± 4.5||5.3 ± 0.4||258 ± 18||26 ± 2.5|
|1000||48 ± 3.7||9 ± 1.4||450 ± 45||34 ± 3.5|
|0||1.5 ± 0.1||0.7 ± 0.2||10 ± 3.5||4.4 ± 1.1|
|1||33 ± 5.7||2.9 ± 0.6||361 ± 32||19 ± 3.4|
|4||23 ± 6.1||3.2 ± 0.7||388 ± 52||21 ± 5.5|
|10||46 ± 13||3.0 ± 0.6||585 ± 101||16 ± 0.3|
|20||61 ± 10||5.9 ± 2.0||1477 ± 172||27 ± 2.4|
aData represent the mean ± SD for 3–5 experiments.
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