FRINK Linked Data Fragments server

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• W2129540903 abstract "Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress. Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress. Oxygen is essential for efficient energy production in aerobes. However, reactive oxygen species generated during this energy production represent a persistent threat to the integrity of the genome. Indeed, oxidative stress is associated with major causes of human mortality including cancer and atherosclerosis.1Halliwell B Gutteridge JMC Free Radicals in Biology and Medicine. Clarendon Press, Oxford1999Google Scholar Reactive oxygen species react with the genomic DNA to cause strand scission, cross-links, or base modifications,2Steenken S Purine bases, nucleosides, and nucleotides: aqueous solution redox chemistry and transformation reactions of their radical reactions and e− and ·OH adducts.Chem Rev. 1989; 89: 503-520Crossref Scopus (1118) Google Scholar with base modifications thought to be the most frequent.3Bernstein C Sex as a response to oxidative DNA damage.in: Halliwell B Aruoma OI DNA and Free Radicals. Ellis Horwood, Chichester1993: 193-210Google Scholar Guanine base exposed either to hydroxyl radical, singlet oxygen, or photodynamic action is hydroxylated at C-8, leading to the formation of 8-hydroxyguanine (8-OHGua),4Kasai H Nishimura S Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents.Nucleic Acids Res. 1984; 12: 2137-2145Crossref PubMed Scopus (880) Google Scholar which is present at a frequency of ∼1 in 105–6 guanine bases in the genomic DNA of various tissues under control conditions.5Kasai H Analysis of a form of oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis.Mutat Res. 1997; 387: 147-163Crossref PubMed Scopus (972) Google Scholar Other modified guanine bases such as 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) may be produced via hydroxyl radicals.6Tudek B Imidazole ring-opened DNA purines and their biological significance.J Biochem Mol Biol (Korea). 2003; 36: 12-19Crossref PubMed Google Scholar 8-OHGua in the genome may cause G to T transversion-type mutations during DNA replication.7Shibutani S Takeshita M Grollman AP Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG.Nature. 1991; 349: 431-434Crossref PubMed Scopus (2027) Google Scholar Thus far, three distinct repair enzymes (OGG1, MUTYH, and MTH1) have been identified to function at different levels to repair or prevent 8-OHGua in the genome. Germ-line mutations in MUTYH have been associated with recessive inheritance of multiple colorectal adenomas.8Al-Tassan N Chmiel N Maynard J Fleming N Livingston A Williams G Hodges A Davies D David S Sampson J Cheadle J Inherited variants of MYH associated with somatic G: C–>T:A mutations in colorectal tumors.Nat Genet. 2002; 30: 227-232Crossref PubMed Scopus (1074) Google Scholar, 9Sieber O Lipton L Crabtree M Heinimann K Fidalgo P Phillips R Bisgaard M Orntoft T Aaltonen L Hodgson S Thomas H Tomlinson I Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH.N Engl J Med. 2003; 348: 791-799Crossref PubMed Scopus (729) Google Scholar Knockout of these repair enzymes in mice have been shown to produce a phenotype of higher cancer incidence.10Tsuzuki T Egashira A Igarashi H Iwakuma T Nakatsuru Y Tominaga Y Kawate H Nakao K Nakamura K Ide F Kura S Nakabeppu Y Katsuki M Ishikawa T Sekiguchi M Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase.Proc Natl Acad Sci USA. 2001; 98: 11456-11461Crossref PubMed Scopus (257) Google Scholar, 11Sakumi K Tominaga Y Furuichi M Xu P Tsuzuki T Sekiguchi M Nakabeppu Y Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene disruption.Cancer Res. 2003; 63: 902-905PubMed Google Scholar, 12Kunisada M Sakumi K Tominaga Y Budiyanto A Ueda M Ichihashi M Nakabeppu Y Nishigori C 8-Oxoguanine formation induced by chronic UVB exposure makes Ogg1 knockout mice susceptible to skin carcinogenesis.Cancer Res. 2005; 65: 6006-6010Crossref PubMed Scopus (110) Google Scholar We have been studying the molecular mechanisms of how reactive oxygen species induce cancer using an iron-mediated rodent renal carcinogenesis model. Our recent genetic analyses identified p16INK4A tumor suppressor gene as a major target gene, and we proposed a hypothesis that fragile sites exist in the genome against oxidative stress.13Tanaka T Iwasa Y Kondo S Hiai H Toyokuni S High incidence of allelic loss on chromosome 5 and inactivation of p15INK4B and p16INK4A tumor suppressor genes in oxystress-induced renal cell carcinoma of rats.Oncogene. 1999; 18: 3793-3797Crossref PubMed Scopus (103) Google Scholar, 14Hiroyasu M Ozeki M Kohda H Echizenya M Tanaka T Hiai H Toyokuni S Specific allelic loss of p16INK4A tumor suppressor gene after weeks of iron-mediated oxidative damage during rat renal carcinogenesis.Am J Pathol. 2002; 160: 419-424Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Thus far, to the best of our knowledge, genome-wide screening for the detection of sites vulnerable to oxidative stress has not been undertaken. This study aimed to establish a method to generate libraries of genomic DNA fragments containing oxidatively modified bases and to use the method to reveal common rules therein. We applied and optimized an immunoprecipitation technique for this purpose. In addition to 8-OHGua, we selected an aldehyde-modified adenine, 1,N6-propanoadenine, which is produced by the reaction of acrolein and adenine.15Kawai Y Furuhata A Toyokuni S Aratani Y Uchida K Formation of acrolein-derived 2′-deoxyadenosine adduct in an iron-induced carcinogenesis model.J Biol Chem. 2003; 278: 50346-50354Crossref PubMed Scopus (62) Google Scholar Acrolein, a major lipid peroxidation product, is chemically reactive and mutagenic and may be one of the second messengers of oxidative stress.16Uchida K Kanematsu M Sakai K Matsuda T Hattori N Mizuno Y Suzuki D Miyata T Noguchi N Niki E Osawa T Protein-bound acrolein: potential markers for oxidative stress.Proc Natl Acad Sci USA. 1998; 95: 4882-4887Crossref PubMed Scopus (546) Google Scholar Previously, we produced and characterized monoclonal antibodies recognizing either 8-hydroxy-2′-deoxyguanosine (8-OHdG)17Toyokuni S Tanaka T Hattori Y Nishiyama Y Ochi H Hiai H Uchida K Osawa T Quantitative immunohistochemical determination of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model.Lab Invest. 1997; 76: 365-374PubMed Google Scholar or 1,N6-propano-2′-deoxyadenosine (acrolein-dA).15Kawai Y Furuhata A Toyokuni S Aratani Y Uchida K Formation of acrolein-derived 2′-deoxyadenosine adduct in an iron-induced carcinogenesis model.J Biol Chem. 2003; 278: 50346-50354Crossref PubMed Scopus (62) Google Scholar Here we examined whether it was possible to specifically immunoprecipitate DNA fragments (DnaIP) containing these two different modified bases and whether there is any principle underlying the distribution of these modified bases in vivo in the genome of renal cortical cells in an oxidative stress (ferric nitrilotriacetate)-induced carcinogenesis model of rodents.18Ebina Y Okada S Hamazaki S Ogino F Li JL Midorikawa O Nephrotoxicity and renal cell carcinoma after use of iron- and aluminum-nitrilotriacetate complexes in rats.J Natl Cancer Inst. 1986; 76: 107-113PubMed Google Scholar, 19Li JL Okada S Hamazaki S Ebina Y Midorikawa O Subacute nephrotoxicity and induction of renal cell carcinoma in mice treated with ferric nitrilotriacetate.Cancer Res. 1987; 47: 1867-1869PubMed Google Scholar, 20Toyokuni S Okada S Hamazaki S Minamiyama Y Yamada Y Liang P Fukunaga Y Midorikawa O Combined histochemical and biochemical analysis of sex hormone dependence of ferric nitrilotriacetate-induced renal lipid peroxidation in ddY mice.Cancer Res. 1990; 50: 5574-5580PubMed Google Scholar, 21Nishiyama Y Suwa H Okamoto K Fukumoto M Hiai H Toyokuni S Low incidence of point mutations in H-, K- and N-ras oncogenes and p53 tumor suppressor gene in renal cell carcinoma and peritoneal mesothelioma of Wistar rats induced by ferric nitrilotriacetate.Jpn J Cancer Res. 1995; 86: 1150-1158Crossref PubMed Scopus (63) Google Scholar, 22Okada S Iron-induced tissue damage and cancer: the role of reactive oxygen free radicals.Pathol Int. 1996; 46: 311-332Crossref PubMed Scopus (168) Google Scholar Male C57BL/6 mice (10 to 12 weeks old; Charles River Japan, Tokyo, Japan) were maintained in a specific pathogen-free environment. Twenty-four animals were divided into three groups of 18, three, and three animals, respectively, consisting of a time course group, untreated control group, and ferric nitrilotriacetate (Fe-NTA) group. Animals of the time course group were used for the selection of timing appropriate for the immunoprecipitation analyses, ie, not too much cellular necrosis but high genomic content of 8-OHdG and acrolein-dA as evaluated by high-performance liquid chromatography and/or immunohistochemistry. The animals received an intraperitoneal injection of 3 mg of iron/kg of Fe-NTA prepared immediately before use23Toyokuni S Uchida K Okamoto K Hattori-Nakakuki Y Hiai H Stadtman ER Formation of 4-hydroxy-2-nonenal-modified proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate.Proc Natl Acad Sci USA. 1994; 91: 2616-2620Crossref PubMed Scopus (334) Google Scholar and were sacrificed at the indicated time after injection (n = 3, untreated, 3, 6, 9, 12, and 24 hours after injection; 6 hours was used for immunoprecipitation). Male OGG1 knockout mice (C57BL/6 background)11Sakumi K Tominaga Y Furuichi M Xu P Tsuzuki T Sekiguchi M Nakabeppu Y Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene disruption.Cancer Res. 2003; 63: 902-905PubMed Google Scholar of the same age were used (n = 3 for each time course group, untreated control, and Fe-NTA groups). The institutional Animal Care and Use Committee of Kyoto University approved all of the animal experimentation protocols. Clone N45.1, which specifically recognizes 8-OHdG,17Toyokuni S Tanaka T Hattori Y Nishiyama Y Ochi H Hiai H Uchida K Osawa T Quantitative immunohistochemical determination of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model.Lab Invest. 1997; 76: 365-374PubMed Google Scholar and clone mAb21, which specifically recognizes acrolein-dA,15Kawai Y Furuhata A Toyokuni S Aratani Y Uchida K Formation of acrolein-derived 2′-deoxyadenosine adduct in an iron-induced carcinogenesis model.J Biol Chem. 2003; 278: 50346-50354Crossref PubMed Scopus (62) Google Scholar were used. A double-stranded 22-bp oligonucleotide containing one 8-OHdG paired with deoxycytidine on the complementary strand was prepared and labeled with fluorescein isothiocyanate (FITC) at the 5′-end of the (+) strand (FITC-5′-GGTGGCCTGACG*CATTCCCCAA-3′; *, 8-OHdG).24Kondo S Toyokuni S Tanaka T Hiai H Onodera H Kasai H Imamura M Overexpression of the hOGG1 gene and high 8-hydroxy-2′-deoxyguanosine (8-OHdG) lyase activity in human colorectal carcinoma: regulation mechanism of the 8-OHdG level in DNA.Clin Cancer Res. 2000; 6: 1394-1400PubMed Google Scholar A double-stranded 22-bp oligonucleotide with the same sequence except without 8-OHdG worked as a control. One hundred fmol of the double-stranded 22-bp oligonucleotide was incubated at 4°C overnight with 0.1 or 100 μg of N45.1 monoclonal antibody in 10 mmol/L phosphate buffer (pH 7.4) in a 50-μl volume, followed by mixing with 50 μl of protein A Sepharose CL-4B (Amersham Pharmacia Biotech, Tokyo, Japan) and incubation on ice for 1 hour. After washing with 100 mmol/L HEPES buffer (pH 8.0), the Sepharose beads were separated by centrifugation, lyophilized, and dissolved in 20 μl of loading buffer [80% formamide, 10 mmol/L NaOH, and 1 mmol/L ethylenediaminetetraacetic acid (EDTA)]. The solution was then denatured by heating at 95°C for 5 minutes. The sample solution was applied to a 20% denaturing polyacrylamide gel containing 8 mol/L urea in 1× Tris-borate EDTA buffer and electrophoresed at 10 W for 30 minutes at room temperature. After electrophoresis, the fluorescence intensity of each band was evaluated using FMBio-100 (TakaraBio, Shiga, Japan). Nuclear genomic DNA was extracted from mouse renal cortical samples by the NaI method (Wako, Osaka, Japan).25Nakae D Mizumoto Y Kobayashi E Noguchi O Konishi Y Improved genomic/nuclear DNA extraction for 8-hydroxydeoxyguanosine analysis of small amounts of rat liver tissue.Cancer Lett. 1995; 97: 233-239Abstract Full Text PDF PubMed Scopus (144) Google Scholar Each solution was saturated with argon gas and supplemented with desferal (final concentration, 0.1 mmol/L) where applicable to prevent further DNA oxidation. To increase the 8-OHdG level without inducing strand breaks, genomic DNA (100 μg/ml; 10 mmol/L Tris-HCl buffer, pH 8.0) in the presence of 5 to 50 mmol/L methylene blue and 0.1 mmol/L desferal was incubated under a 60 W electric bulb (12-cm distance) for 30 minutes as described.26Schneider JE Price S Maidt L Gutteridge JM Floyd RA Methylene blue plus light mediates 8-hydroxy 2′-deoxyguanosine formation in DNA preferentially over strand breakage.Nucleic Acids Res. 1990; 18: 631-635Crossref PubMed Scopus (196) Google Scholar This procedure increased the amounts of 8-OHdG up to ∼1000-fold. The amount of 8-OHdG in DNA was estimated after nuclease P1 and alkaline phosphatase treatment by high-performance liquid chromatography with an electrochemical detector as described17Toyokuni S Tanaka T Hattori Y Nishiyama Y Ochi H Hiai H Uchida K Osawa T Quantitative immunohistochemical determination of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model.Lab Invest. 1997; 76: 365-374PubMed Google Scholar with the following minor modification. Desferal (final concentration, 0.1 mmol/L) was added before nuclease P1 digestion. A pGL3-catalase promoter vector27Toyokuni S Tanaka T Kawaguchi W Fang N Ozeki M Akatsuka S Hiai H Aruoma O Bahorun T Effects of the phenolic contents of Mauritian endemic plant extracts on promoter activities of antioxidant enzymes.Free Radic Res. 2003; 37: 1215-1224Crossref PubMed Scopus (61) Google Scholar was digested with BamHI and HindIII to produce 1.7-, 1.9-, and 4.4-kb fragments (vector-1). Another vector containing a cloned genomic DNA fragment (pCR4BluntTOPO-0078) was digested with EcoRI and KpnI to produce 0.4-, 1.1-, 2.0-, and 3.9-kb fragments (vector-2). DNA fragments from the former vector were treated with methylene blue and light to increase the level of 8-OHdG by ∼500-fold. Equal amounts of both of the DNA fragment preparations were mixed and subjected to DnaIP. Recovered DNA fragments were cloned and identified either by size or by sequencing with an ABI Prism 377 sequencer (Tokyo, Japan). Genomic DNA was digested either with HaeIII (GG/CC) for library construction or with Sau96I (G/GNCC) for amplification in quantitative polymerase chain reaction (PCR) experiments. An aliquot of genomic DNA fragments (20 μg for chromosome mapping; 30 μg for amplification in quantitative PCR experiments) was incubated with each antibody (10 μg of N45.1 or 2 μg of mAb 21) in 10 mmol/L phosphate-buffered saline containing 0.1% bovine serum albumin for 3 hours at 4°C in a 900-μl volume, mixed with 100 μl of Dynabeads M-280 sheep anti-mouse IgG (Dynal, Oslo, Norway) and incubated for another 3 hours. The beads were then washed sequentially with four different buffers (buffer 1: 0.1% sodium deoxycholate, 1% Triton X-100, 1 mmol/L EDTA, 50 mmol/L HEPES-KOH, 140 mmol/L NaCl, pH 7.5; buffer 2: 0.1% sodium deoxycholate, 1% Triton X-100, 1 mmol/L EDTA, 50 mmol/L HEPES-KOH, 500 mmol/L NaCl, pH 7.5; buffer 3: 0.1% sodium deoxycholate, 0.5% Nonidet P-40, 1 mmol/L EDTA, 250 mmol/L LiCl, and 10 mmol/L Tris-HCl, pH 8.0; buffer 4: 1× TE). The beads were incubated with 80 μl of elution buffer (10 mmol/L EDTA, 1% sodium dodecyl sulfate, and 50 mmol/L Tris-HCl, pH 8.0) at 65°C for 10 minutes. This procedure was performed twice. For cloning only, the eluent was treated with calf intestinal alkaline phophatase (TakaraBio). Then, the recovered DNA was digested with proteinase K at 37°C for 1 hour, subjected to phenol-chloroform extraction, and precipitated with ethanol. The amounts of recovered DNA were quantified by the Saran Wrap method using ethidium bromide.28Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York2001Google Scholar Cloning was done with a Zero Blunt TOPO PCR cloning kit for sequencing (Invitrogen, Tokyo, Japan) as suggested by the manufacturer, and the cloned fragments were sequenced with an ABI Prism 377 sequencer. The locations of the cloned fragments on chromosomes were assigned according to the May 2004 assembly of the mouse genome (NCBI m33 build) at UCSC (). RefSeq database () was also used as a reference. Mouse Genome 430, 2.0 Arrays (Affymetrix Inc., Santa Clara, CA) were used. Total RNA was isolated using a RNeasy mini kit (Qiagen, Tokyo, Japan). Pooled RNA from three animals of each group was analyzed. The degree of gene expression was then evaluated with Affymetrix GeneChip operating software (GCOS). Immunoprecipitated DNA fragments were amplified by PCR after ligation with an adaptor (Sau96I, 5′-GNCTGCGGTGA-3′ and 5′-AGCACTCTCCAGCCTCTCACCGCA-3′; underline, complementary sequence; Ligation pack, Nippon Gene, Toyama, Japan) and then subjected to exonuclease I treatment and phenol-chloroform extraction as described.29Lucito R Nakimura M West J Han Y Chin K Jensen K McCombie R Gray J Wigler M Genetic analysis using genomic representations.Proc Natl Acad Sci USA. 1998; 95: 4487-4492Crossref PubMed Scopus (64) Google Scholar The amplified DNA fragments were subjected to quantitation using an ABI 7300 real-time PCR system with the specific primer pairs shown in Table 1. One or two primer pairs were prepared for each gene or intergenic area, and the mean value was used as a result when two primer pairs were selected. The corresponding genomic DNA after Sau96I digestion and amplification was used as a control. The final results were adjusted by the amounts of immunoprecipitated DNA fragments.Table 1Detailed Information about Target Genome Areas in Quantitative PCR AnalysisTarget areaCategoryMapSau96I fragment examined*Alignment on NCBI Mouse Build 33 (and its length). (table continues)Gpx3Highly expressed in the kidney11qB1.354,543,484 to 54,543,937 (454 bp)54,544,840 to 54,545,400 (561 bp)ActbHousekeeping5qG2140,384,767 to 140,385,328 (562 bp)140,389,282 to 140,389,785 (504 bp)Gsta4Response to oxidative stress9qE178,563,608 to 78,564,135 (528 bp)78,567,352 to 78,567,905 (554 bp)TrioHighly expressed at early developmental stage15qB127,892,748 to 27,893,287 (540 bp)Trp53Tumor suppressor (gatekeeper)11qB369,199,039 to 69,199,547 (509 bp)69,201,537 to 69,202,006 (470 bp)Hmox1Response to oxidative stress8qC174,162,238 to 74,162,760 (523 bp)74,166,814 to 74,167,311 (498 bp)AI480653Unknown function16qB230,842,039 to 30,842,562 (524 bp)Pdzk3Secreted protein15qA112,394,886 to 12,395,429 (545 bp)Pax7Developmental stage-specific (silenced in the kidney)4qD3138,218,354 to 138,218,866 (513 bp)138,279,020 to 138,279,573 (554 bp)Cdkn2aTumor suppressor (gatekeeper)4qC487,591,234 to 87,591,826 (593 bp)87,597,610 to 87,598,071 (462 bp)Pax5Developmental stage-specific (silenced in the kidney)4qB143,933,461 to 43,933,976 (516 bp)43,936,346 to 43,936,888 (543 bp)Gabra5Tissue-specific (silenced in the kidney)7qB445,084,988 to 45,085,544 (557 bp)45,106,519 to 45,107,045 (527 bp)Chr10_65Intergenic area10qB5.165,380,121 to 65,380,628 (508 bp)65,381,573 to 65,382,083 (511 bp)Chr15_21Intergenic area15qA221,813,091 to 21,813,675 (585 bp)Chr15_37Intergenic area15qB3.137,211,512 to 37,212,101 (590 bp)Chr15_58Intergenic area15qD158,952,316 to 58,952,844 (529 bp)Chr16_33Mouse mRNAs from GenBank16qB334,035,996 to 34,036,524 (529 bp)Chr16_47Intergenic area16qB547,283,849 to 47,284,395 (547 bp)47,294,616 to 47,295,114 (499 bp)Chr16_58Intergenic area16qC1.258,921,006 to 58,921,534 (529 bp)Chr16_90Mouse mRNAs from GenBank16qC3.391,205,361 to 91,205,886 (526 bp)Primer sequenceProduct size5′-CTGTGCCTTTAGTGTGGGAACT-3′ and 5′-TCACCCATCTCTCCTTCATTCT-3′113 bp5′-TGTAACCAAGCTTGAGACCTGA-3′ and 5′-ATTCACTTATTTGGGGGAGGAT-3′119 bp5′-TGACGTTGACATCCGTAAAGAC-3′ and 5′-GCAGTAATCTCCTTCTGCATCC-3′102 bp5′-TCCAACAAACCAAGAGAAATCC-3′ and 5′-CGACCTCTGAAACAATTCTGGT-3′108 bp5′-CATTGTTGACTGCCTAGCAAAG-3′ and 5′-GGTAAGAAAAGCTCCTGTGTGG-3′141 bp5′-TCTAGCCTCTGGCTCATGGTAT-3′ and 5′-CACTGTCTGATGGAATGCTTGT-3′101 bp5′-TATATTCGTGGGGGTCGATTTC-3′ and 5′-AACAGCCTGTAAGGTGGGAGAG-3′130 bp5′-TAAGCCATAGGGGTTTGTTTGT-3′ and 5′-AGGCTGATATCCGACTGTGACT-3′122 bp5′-ACTTCACCTGGATCCTGTGTCT-3′ and 5′-ACCTGGAGTCTTCCAGTGTGAT-3′142 bp5′-AATCATTCACAGACATCGCTTG-3′ and 5′-CTTCATGAACTCAGCATTCTCG-3′137 bp5′-GGGAGATACCTGACACAGTTCC-3′ and 5′-GCTCAGAACAGCTGCTTTTACA-3′126 bp5′-ATGCTGTCTCCTCCCAACTTTC-3′ and 5′-CTTGCTGGTCAGTTTGAAGTGG-3′126 bp5′-AGGAATGGAGGCTTTGTCAGTC-3′ and 5′-AGAGGGCATATGTTGCCTCATT-3′130 bp5′-CCTTTATCAAACGGGGATTCAC-3′ and 5′-CTCCATTCTTCCGGAAACAAAC-3′128 bp5′-AAGTTGAGGCCTTCGGTTAGTC-3′ and 5′-AGCTAGAGCCCTCTTCTCAAGC-3′138 bp5′-ATGACTTCACCCCGTCACTTTT-3′ and 5′-GGTACCACGCCCAGTATGAAAT-3′121 bp5′-GAAGGAATGGTATCCAGCAGAG-3′ and 5′-TTTACTCTTTCCCACCATGACC-3′114 bp5′-TGTCCACCACAGAACTGCTTTT-3′ and 5′-GACAGTGAGACTTGCCTTGGTG-3′114 bp5′-AGAGCTGGCTCTTCTCCTTTCA-3′ and 5′-ATCCTGGCTCAGGAAACTCAAG-3′130 bp5′-TAAAGCTGTCTCGGTTGGGTTT-3′ and 5′-GTGATGGATTGGTACACGGAAA-3′104 bp5′-GTGTTTCTGACATGTGCCTTTC-3′ and 5′-CTGAAATATTTGCCACACGTTC-3′101 bp5′-GCTAGAGCAAGAGCAAAACCAT-3′ and 5′-GAAGCCATGCTTCTAAATGAGG-3′128 bp5′-AAGTTGTCAAGGGAAGGAAGC-3′ and 5′-CCACTATTGCATCTTCGGGTA-3′102 bp5′-TGTGGACTCACAGAATGCCTTA-3′ and 5′-GACCAGTGGATTCCATGATTGT-3′125 bp5′-TCTCTGTTCTTCTGCAGCATCC-3′ and 5′-GAACTGCTGGAGTATGGCTCCT-3′141 bp5′-TTTTTCAGCACGGGTACTTGAG-3′ and 5′-GGCAGCAGTATCAAGCAGAGAC-3′147 bp5′-GTACTGAGGATGCCTGTGTTGC-3′ and 5′-CATGAGCCCAGACAGGTAACAG-3′115 bp5′-AGGCAGAGGTTTTGACTTGGAG-3′ and 5′-TCGACAAACTGTAATGCCAACC-3′130 bp5′-CCAATTGGAGCTAACAGAAACC-3′ and 5′-AGCTGGTCAACTGCCTACTCTC-3′113 bp5′-TGTTTTCCGGAGTTTCAGTTCA-3′ and 5′-ACCCTTGTTTCTCCCATGTTGT-3′133 bp5′-CTAGCATGCCACTTCCATGTTC-3′ and 5′-TGCGGTTCTTTCACTTGTTGTT-3′107 bp* Alignment on NCBI Mouse Build 33 (and its length). (table continues) Open table in a new tab A touch preparation of renal cortex was obtained as previously described. More than 90% were nuclei of proximal tubular cells.30Toyokuni S Iwasa Y Kondo S Tanaka T Ochi H Hiai H Intranuclear distribution of 8-hydroxy-2′-deoxyguanosine. An immunocytochemical study.J Histochem Cytochem. 1999; 47: 833-836Crossref PubMed Scopus (33) Google Scholar The specimens were subjected to fluorescent in situ hybridization analysis with chromosome painting probes according to the manufacturer's instructions (dual-color biotin/Texas Red-FITC; Cambio, Cambridge, UK) and were observed with a confocal laser microscope (Fluoview; Olympus, Osaka, Japan). The center of gravity of the nucleus and that of the chromosome were measured to assign the relative radial location. Histological and immunohistochemical analyses were performed as previously described.15Kawai Y Furuhata A Toyokuni S Aratani Y Uchida K Formation of acrolein-derived 2′-deoxyadenosine adduct in an iron-induced carcinogenesis model.J Biol Chem. 2003; 278: 50346-50354Crossref PubMed Scopus (62) Google Scholar, 17Toyokuni S Tanaka T Hattori Y Nishiyama Y Ochi H Hiai H Uchida K Osawa T Quantitative immunohistochemical determination of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model.Lab Invest. 1997; 76: 365-374PubMed Google Scholar Neutral formalin-fixed paraffin-embedded sections were used with the avidin-biotin complex method (primary antibody concentration: N45.1, 10 μg/ml; mAb 21, 1.3 μg/ml). Three registered pathologists (T.S., T.T., and S.T.) conducted all of the pathological analyses. Statistical analyses were performed with an unpaired t-test, which was modified for unequal variances when necessary, χ2Steenken S Purine bases, nucleosides, and nucleotides: aqueous solution redox chemistry and transformation reactions of their radical reactions and e− and ·OH adducts.Chem Rev. 1989; 89: 503-520Crossref Scopus (1118) Google Scholar test, Kolmogorov-Smirnov test,31Stephens MA Use of the Kolmogorov-Smirnov, Cramer-Von Mises and related statistics without extensive tables.J Roy Stat Soc B. 1970; 32: 115-122Google Scholar or one-way analysis of variance. P < 0.05 was considered to be statistically significant. To test our strategy (Figure 1A), double-stranded 22-mer oligo-DNA fragments containing a single 8-OHdG were synthesized and subjected to DnaIP. Although oligo-DNA fragments were bound to the beads in the absence of antibody (Figure 1B, large arrowhead), the inclusion of a small amount of antibody was enough to dramatically decrease this nonspecific binding (Figure 1B, small arrowhead). In the presence of specific antibody against 8-OHdG (clone N45.1), only DNA fragments containing 8-OHdG, but not those of the same sequence without 8-OHdG, were immunoprecipitated (Figure 1B). Then, genomic DNA extracted from mouse cortical kidney was used after digestion with HaeIII (GG/CC). The average size of the DNA fragments was ∼1 kbp. N45.1 but not control antibody of the same isotype (IgG1) immunoprecipitated the DNA fragments. On the contrary, an excess of antibody decreased the final recovery of DNA fragments (Figure 1, C and D) probably because of the limited binding capacity of the beads for IgG. Similar results to those in Figure 1, C and D, were obtained for acrolein-dA and its specific monoclonal antibody (data not shown). The amounts of immunoprecipitated DNA was basically in proportion with the amounts of loaded DNA (Figure 1E) or the fraction of 8-OHdG (Figure 1F). When equal amounts of enzyme-digested double-stranded DNA vector-1 and −2 (only vector-1 was incubated with methylene blue under light to increase the 8-OHdG level by ∼500-fold) were mixed and subjected to DnaIP, 20 fragments of 21 (95.2%) obtained were from vector-1. An oxidative stress-induced mouse model of renal carcinogenesis was used.19Li JL Okada S Hamazaki S Ebina Y Midorikawa O Subacute nephrotoxicity and induction of renal cell carcinoma in mice treated with ferric nitrilotriacetate.Cancer Res. 1987; 47: 1867-1869PubMed Google Scholar In this model, the Fenton reaction occurs in the renal proximal tubules after an intraperitoneal injection of ferric nitrilotriacetate (Fe-NTA).20Toyokuni S Okada S Hamazaki S Minamiyama Y Yamada Y Liang P Fukunaga Y Midorikawa O Combined histochemical and biochemical analysis of sex hormone dependence of ferric nitrilotriacetate-i" @default.
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• W2129540903 date "2006-10-01" @default.
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• W2129540903 title "Contrasting Genome-Wide Distribution of 8-Hydroxyguanine and Acrolein-Modified Adenine during Oxidative Stress-Induced Renal Carcinogenesis" @default.
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• W2129540903 doi "https://doi.org/10.2353/ajpath.2006.051280" @default.
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