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Comprehensive Assessment of Oxidatively Induced Modifications of DNA in a Rat Model of Human Wilson's Disease

2015; Elsevier BV; Volume: 15; Issue: 3 Linguagem: Inglês

10.1074/mcp.m115.052696

ISSN

1535-9484

Autores

Yang Yu, Candace R. Guerrero, Shuo Liu, Nicholas J. Amato, Yogeshwar Sharma, Sanjeev Gupta, Yinsheng Wang,

Tópico(s)

Drug Transport and Resistance Mechanisms

Resumo

Defective copper excretion from hepatocytes in Wilson's disease causes accumulation of copper ions with increased generation of reactive oxygen species via the Fenton-type reaction. Here we developed a nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry coupled with the isotope-dilution method for the simultaneous quantification of oxidatively induced DNA modifications. This method enabled measurement, in microgram quantities of DNA, of four oxidative stress-induced lesions, including direct ROS-induced purine cyclonucleosides (cPus) and two exocyclic adducts induced by byproducts of lipid peroxidation, i.e. 1,N6-etheno-2′-deoxyadenosine (εdA) and 1,N2-etheno-2′-deoxyguanosine (εdG). Analysis of liver tissues of Long-Evans Cinnamon rats, which constitute an animal model of human Wilson's disease, and their healthy counterparts [i.e. Long-Evans Agouti rats] showed significantly higher levels of all four DNA lesions in Long-Evans Cinnamon than Long-Evans Agouti rats. Moreover, cPus were present at much higher levels than εdA and εdG lesions. In contrast, the level of 5-hydroxymethyl-2′-deoxycytidine (5-HmdC), an oxidation product of 5-methyl-2′-deoxycytidine (5-mdC), was markedly lower in the liver tissues of Long-Evans Cinnamon than Long-Evans Agouti rats, though no differences were observed for the levels of 5-mdC. In vitro biochemical assay showed that Cu2+ ions could directly inhibit the activity of Tet enzymes. Together, these results suggest that aberrant copper accumulation may perturb genomic stability by elevating oxidatively induced DNA lesions, and by altering epigenetic pathways of gene regulation. Defective copper excretion from hepatocytes in Wilson's disease causes accumulation of copper ions with increased generation of reactive oxygen species via the Fenton-type reaction. Here we developed a nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry coupled with the isotope-dilution method for the simultaneous quantification of oxidatively induced DNA modifications. This method enabled measurement, in microgram quantities of DNA, of four oxidative stress-induced lesions, including direct ROS-induced purine cyclonucleosides (cPus) and two exocyclic adducts induced by byproducts of lipid peroxidation, i.e. 1,N6-etheno-2′-deoxyadenosine (εdA) and 1,N2-etheno-2′-deoxyguanosine (εdG). Analysis of liver tissues of Long-Evans Cinnamon rats, which constitute an animal model of human Wilson's disease, and their healthy counterparts [i.e. Long-Evans Agouti rats] showed significantly higher levels of all four DNA lesions in Long-Evans Cinnamon than Long-Evans Agouti rats. Moreover, cPus were present at much higher levels than εdA and εdG lesions. In contrast, the level of 5-hydroxymethyl-2′-deoxycytidine (5-HmdC), an oxidation product of 5-methyl-2′-deoxycytidine (5-mdC), was markedly lower in the liver tissues of Long-Evans Cinnamon than Long-Evans Agouti rats, though no differences were observed for the levels of 5-mdC. In vitro biochemical assay showed that Cu2+ ions could directly inhibit the activity of Tet enzymes. Together, these results suggest that aberrant copper accumulation may perturb genomic stability by elevating oxidatively induced DNA lesions, and by altering epigenetic pathways of gene regulation. Many endogenous and exogenous chemical events may produce DNA damage, including assault from reactive oxygen species (ROS) 1The abbreviations used are:ROSreactive oxygen speciescPu8,5′-cyclopurine-2′-deoxynucleosideεdA1,N6-etheno-2′-deoxyadenosineεdG1,N2-etheno-2′-deoxyguanosineLPOlipid peroxidation2-OG2-oxoglutarateTETten-eleven translocationSICselected ion chromatogram. (1.Finkel T. Holbrook N.J. Oxidants, oxidative stress and the biology of ageing.Nature. 2000; 408: 239-247Crossref PubMed Scopus (7298) Google Scholar, 2.Friedberg E.C. Walker G.C. Siede W. Wood R.D. Schultz R.A. Ellenberger T. DNA Repair and Mutagenesis. ASM Press, Washington, D.C.2006Google Scholar). ROS are routinely generated in cells as a consequence of metabolic activity and exposure to various environmental agents. Under physiological conditions, transition metal ion-mediated Fenton reaction constitutes a major endogenous source of ROS, and aberrant accumulation of transition metal ions may elicit deleterious effects on cells. Elevated ROS could lead to DNA damage directly, or indirectly via byproducts of lipid peroxidation (LPO). In this vein, hydroxyl radical can directly react with DNA to yield a myriad of DNA lesions, including the bulky 8,5′-cyclopurine-2′-deoxynucleosides (cPu) (3.Brooks P.J. The case for 8,5′-cyclopurine-2′-deoxynucleosides as endogenous DNA lesions that cause neurodegeneration in xeroderma pigmentosum.Neuroscience. 2007; 145: 1407-1417Crossref PubMed Scopus (97) Google Scholar, 4.Wang Y. Bulky DNA lesions induced by reactive oxygen species.Chem. Res. Toxicol. 2008; 21: 276-281Crossref PubMed Scopus (135) Google Scholar). 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In this context, Fe2+- and 2-oxoglutarate (2-OG)-dependent ten-eleven translocation (TET) family dioxygenases were recently found to be involved in sequential oxidation of 5-methyl-2′-deoxycytidine (5-mdC) to yield 5-hydroxymethyl-2′-deoxycytidine (5-HmdC), 5-formyl-2′-deoxycytidine (5-FodC), and 5-carboxyl-2′-deoxycytidine (5-CadC) (15.Pfaffeneder T. Hackner B. Truss M. Muenzel M. Mueller M. Deiml C.A. Hagemeier C. Carell T. The discovery of 5-formylcytosine in embryonic stem cell DNA.Angew. Chem. Int. Ed. 2011; 50: 7008-7012Crossref PubMed Scopus (391) Google Scholar, 16.He Y.F. Li B.Z. Li Z. Liu P. Wang Y. Tang Q. Ding J. Jia Y. Chen Z. Li L. Sun Y. Li X. Dai Q. Song C.X. Zhang K. He C. Xu G.L. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.Science. 2011; 333: 1303-1307Crossref PubMed Scopus (2014) Google Scholar, 17.Ito S. D'Alessio A.C. Taranova O.V. Hong K. Sowers L.C. Zhang Y. 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Subsequent excision of 5-FodC and 5-CadC from DNA by thymine DNA glycosylase (TDG), coupled with restoration of an unmethylated 2′-deoxycytidine by base-excision repair (BER) pathway, may facilitate active DNA cytosine demethylation in mammals (16.He Y.F. Li B.Z. Li Z. Liu P. Wang Y. Tang Q. Ding J. Jia Y. Chen Z. Li L. Sun Y. Li X. Dai Q. Song C.X. Zhang K. He C. Xu G.L. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.Science. 2011; 333: 1303-1307Crossref PubMed Scopus (2014) Google Scholar, 21.Maiti A. Drohat A.C. Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites.J. Biol. Chem. 2011; 286: 35334-35338Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). In addition, 5-hmdC could be recognized by some specific cellular proteins (22.Mellén M. Ayata P. Dewell S. Kriaucionis S. Heintz N. MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system.Cell. 2012; 151: 1417-1430Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 23.Yildirim O. Li R. Hung J.H. Chen P.B. Dong X. Ee L.S. Weng Z. Rando O.J. Fazzio T.G. Mbd3/NURD complex regulates expression of 5-hydroxymethylcytosine marked genes in embryonic stem cells.Cell. 2011; 147: 1498-1510Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 24.Spruijt C.G. Gnerlich F. Smits A.H. Pfaffeneder T. Jansen P.W. Bauer C. Münzel M. Wagner M. Müller M. Khan F. Eberl H.C. Mensinga A. Brinkman A.B. Lephikov K. Müller U. Walter J. Boelens R. van Ingen H. Leonhardt H. Carell T. Vermeulen M. Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives.Cell. 2013; 152: 1146-1159Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar, 25.Frauer C. Hoffmann T. Bultmann S. Casa V. Cardoso M.C. Antes I. Leonhardt H. Recognition of 5-hydroxymethylcytosine by the Uhrf1 SRA domain.PLoS ONE. 2011; 6: e21306Crossref PubMed Scopus (151) Google Scholar), suggesting that the oxidized derivatives of 5-mdC may also serve as epigenetic marks on their own. Transition metal ion homeostasis is essential for normal cellular function and excess accumulation of metal ions, such as iron or copper, may result in diseases. In this vein, Wilson's disease (WD) is an autosomal recessive genetic disorder arising from mutations in the P-type ATPase gene, ATP7B, and patients suffering from this disease are manifested by defective excretion of copper ions into bile (26.Ala A. Walker A.P. Ashkan K. Dooley J.S. Schilsky M.L. Wilson's disease.Lancet. 2007; 369: 397-408Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar, 27.Roberts E.A. Schilsky M.L. Diagnosis and treatment of Wilson disease: An update.Hepatology. 2008; 47: 2089-2111Crossref PubMed Scopus (936) Google Scholar). This leads to accumulation of copper in the body with progressive damage in liver, brain and kidneys (26.Ala A. Walker A.P. Ashkan K. Dooley J.S. Schilsky M.L. Wilson's disease.Lancet. 2007; 369: 397-408Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar, 27.Roberts E.A. Schilsky M.L. Diagnosis and treatment of Wilson disease: An update.Hepatology. 2008; 47: 2089-2111Crossref PubMed Scopus (936) Google Scholar). The Long-Evans Cinnamon (LEC) rat, which was found in a colony of healthy Long-Evans Agouti (LEA) rat, harbors a partial deletion at the 3′ end of the Atp7b gene and shares many clinical attributes of Wilson's disease in humans, including excess copper accumulation in the liver along with spontaneous acute liver failure and progressive liver damage with extensive hepatic cholangiofibrosis (28.Li Y. Togashi Y. Sato S. Emoto T. Kang J.H. Takeichi N. Kobayashi H. Kojima Y. Une Y. Uchino J. Spontaneous hepatic copper accumulation in Long-Evans Cinnamon rats with hereditary hepatitis. A model of Wilson's disease.J. Clin. Invest. 1991; 87: 1858-1861Crossref PubMed Scopus (287) Google Scholar). Therefore, the LEC rat serves as an excellent animal model for studying the pathophysiology of human Wilson's disease. Increased copper content, and elevated levels of ROS and oxidative DNA damage have been observed in liver tissues of LEC rats (10.Wang J. Yuan B. Guerrero C. Bahde R. Gupta S. Wang Y. Quantification of oxidative DNA lesions in tissues of Long-Evans Cinnamon rats by capillary high-performance liquid chromatography-tandem mass spectrometry coupled with stable isotope-dilution method.Anal. Chem. 2011; 83: 2201-2209Crossref PubMed Scopus (91) Google Scholar, 29.Nair J. Strand S. Frank N. Knauft J. Wesch H. Galle P.R. Bartsch H. Apoptosis and age-dependant induction of nuclear and mitochondrial etheno-DNA adducts in Long-Evans Cinnamon (LEC) rats: enhanced DNA damage by dietary curcumin upon copper accumulation.Carcinogenesis. 2005; 26: 1307-1315Crossref PubMed Scopus (75) Google Scholar). However, it remains unclear whether abnormal copper accumulation perturbs epigenetic signaling by modulating the formation of 5-HmdC. In light of our previous finding that 5-HmdC could be induced from oxidation of 5-mdC in synthetic duplex DNA by Fenton-type reagent (Cu2+/H2O2/ascorbate) in vitro (30.Cao H. Wang Y. Quantification of oxidative single-base and intrastrand cross-link lesions in unmethylated and CpG-methylated DNA induced by Fenton-type reagents.Nucleic Acids Res. 2007; 35: 4833-4844Crossref PubMed Scopus (69) Google Scholar), we reasoned that increased copper accumulation might also elicit higher levels of 5-HmdC in vivo. On the other hand, the Jumonji C domain-containing histone demethylases (JHDMs), which are also Fe2+- and 2-OG-dependent dioxygenases, were previously shown to be perturbed by exposure to carcinogenic heavy metals, including nickel, chromium, and possibly arsenic (31.Chen H. Giri N.C. Zhang R. Yamane K. Zhang Y. Maroney M. Costa M. Nickel ions inhibit histone demethylase JMJD1A and DNA repair enzyme ABH2 by replacing the ferrous iron in the catalytic centers.J. Biol. Chem. 2010; 285: 7374-7383Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 32.Chervona Y. Arita A. Costa M. Carcinogenic metals and the epigenome: understanding the effect of nickel, arsenic, and chromium.Metallomics. 2012; 4: 619-627Crossref PubMed Scopus (187) Google Scholar, 33.Chervona Y. Costa M. The control of histone methylation and gene expression by oxidative stress, hypoxia, and metals.Free Radic. Biol. Med. 2012; 53: 1041-1047Crossref PubMed Scopus (123) Google Scholar). The replacement of iron in the active site of JHDMs by nickel, and depletion of ascorbic acid, a crucial cofactor of these enzymes, from chromium or arsenic exposure, could attenuate the demethylase activity of JHDMs in cells (31.Chen H. Giri N.C. Zhang R. Yamane K. Zhang Y. Maroney M. Costa M. Nickel ions inhibit histone demethylase JMJD1A and DNA repair enzyme ABH2 by replacing the ferrous iron in the catalytic centers.J. Biol. Chem. 2010; 285: 7374-7383Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 32.Chervona Y. Arita A. Costa M. Carcinogenic metals and the epigenome: understanding the effect of nickel, arsenic, and chromium.Metallomics. 2012; 4: 619-627Crossref PubMed Scopus (187) Google Scholar, 33.Chervona Y. Costa M. The control of histone methylation and gene expression by oxidative stress, hypoxia, and metals.Free Radic. Biol. Med. 2012; 53: 1041-1047Crossref PubMed Scopus (123) Google Scholar). Likewise, other Fe2+- and 2-OG-dependent dioxygenases, including hypoxia inducible factor (HIF)-prolyl hydroxylase PHD2 and DNA repair enzymes ALKBH2/3, could also be inhibited by nickel ion (31.Chen H. Giri N.C. Zhang R. Yamane K. Zhang Y. Maroney M. Costa M. Nickel ions inhibit histone demethylase JMJD1A and DNA repair enzyme ABH2 by replacing the ferrous iron in the catalytic centers.J. Biol. Chem. 2010; 285: 7374-7383Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 34.Chen H. Costa M. Iron- and 2-oxoglutarate-dependent Dioxygenases: an emerging group of molecular targets for nickel toxicity and carcinogenicity.Biometals. 2009; 22: 191-196Crossref PubMed Scopus (63) Google Scholar). Therefore, accumulation of copper in Wilson's disease may compromise enzymatic activities of Tet proteins in vivo, thereby resulting in lower 5-HmdC levels. Herein, we comprehensively assessed the effect of abnormal copper accumulation on oxidatively induced DNA modifications in liver and brain tissues of LEC and LEA rats by LC-MS/MS combined with the stable isotope-dilution technique. The goal was to gain a better understanding of genetic and epigenetic alterations arising from aberrant copper accumulation. All chemicals and enzymes, unless otherwise specified, were purchased from Sigma-Aldrich (St. Louis, MO) and New England Biolabs (Ipswich, WA), respectively. Stable isotope-labeled compounds were obtained from Cambridge Isotope Laboratories (Cambridge, MA). Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) hydrochloride was from Tocris Bioscience (Ellisville, MO). LEA and LEC rats (14–16 week old) were bred in a Core Facility at Einstein as described previously (10.Wang J. Yuan B. Guerrero C. Bahde R. Gupta S. Wang Y. Quantification of oxidative DNA lesions in tissues of Long-Evans Cinnamon rats by capillary high-performance liquid chromatography-tandem mass spectrometry coupled with stable isotope-dilution method.Anal. Chem. 2011; 83: 2201-2209Crossref PubMed Scopus (91) Google Scholar). The stable isotope-labeled S-cdA, S-cdG and 5-HmdC were synthesized previously (10.Wang J. Yuan B. Guerrero C. Bahde R. Gupta S. Wang Y. Quantification of oxidative DNA lesions in tissues of Long-Evans Cinnamon rats by capillary high-performance liquid chromatography-tandem mass spectrometry coupled with stable isotope-dilution method.Anal. Chem. 2011; 83: 2201-2209Crossref PubMed Scopus (91) Google Scholar, 30.Cao H. Wang Y. Quantification of oxidative single-base and intrastrand cross-link lesions in unmethylated and CpG-methylated DNA induced by Fenton-type reagents.Nucleic Acids Res. 2007; 35: 4833-4844Crossref PubMed Scopus (69) Google Scholar). The εdA and εdG as well as their stable isotope-labeled counterparts were synthesized following procedures published previously (35.Chen H.J. Chiang L.C. Tseng M.C. Zhang L.L. Ni J. Chung F.L. Detection and quantification of 1,N6-ethenoadenine in human placental DNA by mass spectrometry.Chem. Res. Toxicol. 1999; 12: 1119-1126Crossref PubMed Scopus (62) Google Scholar, 36.Garcia C.C. Freitas F.P. Di Mascio P. Medeiros M.H. Ultrasensitive simultaneous quantification of 1,N2-etheno-2′-deoxyguanosine and 1,N2-propano-2′-deoxyguanosine in DNA by an online liquid chromatography-electrospray tandem mass spectrometry assay.Chem. Res. Toxicol. 2010; 23: 1245-1255Crossref PubMed Scopus (20) Google Scholar). Genomic DNA was extracted from rat tissues by using a high-salt method (10.Wang J. Yuan B. Guerrero C. Bahde R. Gupta S. Wang Y. Quantification of oxidative DNA lesions in tissues of Long-Evans Cinnamon rats by capillary high-performance liquid chromatography-tandem mass spectrometry coupled with stable isotope-dilution method.Anal. Chem. 2011; 83: 2201-2209Crossref PubMed Scopus (91) Google Scholar). The liver and brain tissues of LEA/LEC rats were grounded into fine powders under liquid nitrogen in a mortar. A 100-μl lysis buffer, containing 20 mm Tris (pH 8.1), 20 mm EDTA, 400 mm NaCl, 1% SDS (w/v) and 10–15 μl of proteinase K (20 mg/ml), was added to the powdered tissue with incubation in a water bath at 55 °C overnight. Saturated NaCl solution (0.5 volume) was subsequently added to the mixture, which was vortexed for 1 min and incubated at 55 °C for another 15 min. The resulting mixture was centrifuged at 13,000 rpm for 30 min. Nucleic acids were precipitated from the supernatant by ethanol. The RNA in the nucleic acid mixture was digested with 3 μl RNase A (10 mg/ml) and 2 μl RNase T1 (25 units/μl) at 37 °C overnight, followed by extraction with an equal volume of chloroform/isoamyl alcohol (24:1, v/v). The DNA was precipitated from the aqueous layer by ethanol, re-dissolved in water and quantified by UV spectrophotometry. The DNA sample (10 μg) was digested with 1 unit of nuclease P1 and 0.00125 unit of phosphodiesterase II in a 15-μl buffer containing 300 mm sodium acetate (pH 5.6), 10 mm ZnCl2 and 2.5 nmol of EHNA, which served as an inhibitor of adenine deaminase to minimize deamination of dA to 2′-deoxyinosine (10.Wang J. Yuan B. Guerrero C. Bahde R. Gupta S. Wang Y. Quantification of oxidative DNA lesions in tissues of Long-Evans Cinnamon rats by capillary high-performance liquid chromatography-tandem mass spectrometry coupled with stable isotope-dilution method.Anal. Chem. 2011; 83: 2201-2209Crossref PubMed Scopus (91) Google Scholar). After incubation at 37 °C for 48 h, to the mixture were added 1.0 unit of alkaline phosphatase, 0.0025 unit of phosphodiesterase I, and 20 μl of buffer containing 500 mm Tris-HCl (pH 8.9). After a 2-hr incubation at 37 °C, the digestion mixture was neutralized with 1 m formic acid, and uniformly 15N-labeled S-cdA, S-cdG, εdA and εdG internal standards were added to the mixture. The enzymes in the digestion mixture were removed by extraction with chloroform/isoamyl alcohol (24:1, v/v), and the resulting aqueous layer was subjected to off-line HPLC enrichment. For quantification of 5-HmdC and 5-mdC, [1,3-15N2-2′-D]-5-HmdC and [1′,2′,3′,4′,5′-13C5]-5-mdC were added to the enzymatic digestion mixture of 50 ng of genomic DNA. The enzymes in digestion mixture were again removed by extraction with chloroform/isoamyl alcohol (24:1, v/v), and the resulting aqueous layer was subjected directly to LC-MS/MS analysis on an LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific). The HPLC separation was performed on a Beckman HPLC system with pump module 125 and UV detector module 126. An Alltima HP-C18 column (4.6 × 250 mm, 5 μm in particle size, 300 Å in pore size, Grace Davison, Deerfield, IL) was used to enrich oxidatively induced DNA lesions from the aforementioned nucleoside mixture. The mobile phases were 10 mm ammonium formate (Solution A) and methanol (Solution B). A gradient of 42 min 0% B, 1 min 0–2% B, 17 min 2% B, 1 min 2–5% B, 9 min 5% B, 10 min 5–13% B, 20 min 13% B, and 50 min 13–60% B was used, and the flow rate was 1 ml/min. The HPLC fractions containing S-cdG (33.5–38.0 min), S-cdA (66.0–70.0 min), and εdA + εdG (81.5–87.5 min) were pooled individually and dried in a Speed-vac. The dried samples were then reconstituted in water for NanoLC-NSI/MS2 analysis. The NanoLC-NSI-MS/MS analysis was conducted on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific) equipped with a nano-electrospray ionization source and coupled with an EASY nLC II (Thermo Fisher Scientific). HPLC separation was carried out using a homemade trapping column (150 μm × 40 mm) and an analytical column (75 μm × 200 mm), both packed with Magic C18 AQ (200 Å, 5 μm, Michrom BioResources, Auburn, CA). Mobile phase A was 0.1% formic acid in H2O and mobile phase B was 0.1% formic acid in acetonitrile. Initially, the sample was loaded onto the trapping column with mobile phase A at a flow rate of 2.5 μl/min. The analytes were eluted using a 40-min linear gradient of 0–40% mobile phase B at a flow rate of 300 nL/min. The TSQ Vantage mass spectrometer was operated in the positive-ion mode, where the spray voltage was 1.8 kV and the temperature for ion-transport tube was 275 °C. The instrument was set up in multiple-reaction monitoring (MRM) mode, with MRM transitions being listed in supplemental Table S1. The sensitivities for detecting all modified nucleosides were optimized by varying the collision energy (supplemental Table S1). The amounts of oxidative stress-induced DNA modifications (in moles) in nucleoside mixtures were calculated from area ratios of peaks found in selected-ion chromatograms (SICs) for the analytes over their corresponding isotope-labeled standards, the amounts of the labeled standards added (in moles) and the calibration curves (Fig. S2). The final levels of the modified nucleosides, in terms of the numbers of modified nucleosides per 107 nucleosides, were calculated by dividing the amounts of individual modified nucleosides with the total amount of nucleosides (in moles) in the digested DNA. The LC-MS3 quantification of 5-HmdC was conducted on an LTQ linear ion trap mass spectrometer coupled with an Agilent 1200 capillary HPLC pump, as described previously (37.Liu S. Wang J. Su Y. Guerrero C. Zeng Y. Mitra D. Brooks P.J. Fisher D.E. Song H. Wang Y. Quantitative assessment of Tet-induced oxidation products of 5-methylcytosine in cellular and tissue DNA.Nucleic Acids Res. 2013; 41: 6421-6429Crossref PubMed Scopus (111) Google Scholar). The LC-MS2 measurement of 5-mdC was performed on the same instrument. The HPLC separation was conducted using a 0.5 × 250 mm Zorbax SB-C18 column (5 μm in particle size, Agilent Technologies). A solution of 2 mm ammonium bicarbonate in water (pH 7.0, solution A) and methanol (solution B) were employed as mobile phases. A gradient of 5 min 0–20% B and 25 min 20–70% B was used for separating analytes, and the flow rate was 8.0 μl/min. The voltage for electrospray was 5.0 kV, and the ion-transport tube was maintained at a temperature of 275 °C. The isolation width for precursor ion selection was 3 m/z units, the normalized collision energy was 35, the activation Q was 0.25, and the activation time was 30 ms. The levels of 5-HmdC and 5-mdC were calculated with the method described above for the oxidative stress-induced DNA lesions. The Tet1-mediated 5-mdC oxidation assay was conducted with the use of the 5mC Tet1 Oxidation Kit (Wisegene, Chicago, IL) and a 5-mdC-containing duplex DNA, d(AGCTC(5-mdC)GGTCA)/d(GTGACCGGAGCTG), following procedures reported previously (38.Fu L. Guerrero C.R. Zhong N. Amato N.J. Liu Y. Liu S. Cai Q. Ji D. Jin S.G. Niedernhofer L.J. Pfeifer G.P. Xu G.L. Wang Y. Tet-mediated formation of 5-hydroxymethylcytosine in RNA.J. Am. Chem. Soc. 2014; 136: 11582-11585Crossref PubMed Scopus (210) Google Scholar) with slight modifications. Briefly, 20 pmol of the aforementioned duplex DNA was incubated with 0.4 μl of mouse Tet1 protein, along with a reaction buffer containing 50 mm HEPES (pH 8.0), 100 mm sodium chloride, 2 mm ascorbic acid, 1 mm 2-oxoglutarate, 1 mm ATP, 1 mm DTT, along with 75 μm ammonium iron(II) sulfate and various concentrations of copper sulfate (at Cu2+/Fe2+ molar ratios of 0, 0.25, 0.50, 1.0, 2.0 and 5.0) in a total volume of 50 μl. After incubation at 37 °C for 10 min, the reaction mixtures were immediately frozen on dry ice. The enzyme in the mixture was removed using chloroform extraction. The resulting aqueous layer was subjected directly to LC-MS and MS2 on an LTQ linear ion trap mass spectrometer to identify reaction products and to quantify conversions of 5-mdC to its oxidation products. A 0.5 × 250 mm Zorbax SB-C18 column was employed for the separation and the flow rate was 8.0 μl/min. 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP, pH adjusted to 7.0 with triethylamine, solution A) and methanol (solution B) were employed as mobile phases, and a gradient of 5 min of 0–20% B and 35 min of 20–40% B was used. The voltage for electrospray was 4.0 kV and the ion-transport tube of the mass spectrometer was set at 300 °C. The MS2 and higher-resolution "ultra-zoom-scan" MS were acquired for the [M - 3H]3− ions of the initial 11mer

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