title: Quantitative detection of 4-hydroxyequilenin-DNA adducts in mammalian cells using an immunoassay with a novel monoclonal antibody.
abstract: Estrogen-DNA adducts are potential biomarkers for assessing the risk and development of estrogen-associated cancers. 4-Hydroxyequilenin (4-OHEN) and 4-hydroxyequilin (4-OHEQ), the metabolites of equine estrogens present in common hormone replacement therapy (HRT) formulations, are capable of producing bulky 4-OHEN-DNA adducts. Although the formation of 4-OHEN-DNA adducts has been reported, their quantitative detection in mammalian cells has not been done. To quantify such DNA adducts, we generated a novel monoclonal antibody (4OHEN-1) specific for 4-OHEN-DNA adducts. The primary epitope recognized is one type of stereoisomers of 4-OHEN-dA adducts and of 4-OHEN-dC adducts in DNA. An immunoassay with 4OHEN-1 revealed a linear dose-response between known amounts of 4-OHEN-DNA adducts and the antibody binding to those adducts, with a detection limit of approximately five adducts/10(8) bases in 1 microg DNA sample. In human breast cancer cells, the quantitative immunoassay revealed that 4-OHEN produces five times more 4-OHEN-DNA adducts than does 4-OHEQ. Moreover, in a mouse model for HRT, oral administration of Premarin increased the levels of 4-OHEN-DNA adducts in various tissues, including the uterus and ovaries, in a time-dependent manner. Thus, we succeeded in establishing a novel immunoassay for quantitative detection of 4-OHEN-DNA adducts in mammalian cells.
authors.affiliation: Radioisotope Research Center, Department of Neurology and Medical Genetics Research Center, Nara Medical University School of Medicine, Kashihara, Nara 634-8521, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Okahashi
authors.forename: Takaaki
authors.initials: T
authors.lastname: Iwamoto
authors.forename: Naomi
authors.initials: N
authors.lastname: Suzuki
authors.forename: Shinya
authors.initials: S
authors.lastname: Shibutani
authors.forename: Shigeki
authors.initials: S
authors.lastname: Sugiura
authors.forename: Shinji
authors.initials: S
authors.lastname: Itoh
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Nishiwaki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ueno
authors.forename: Toshio
authors.initials: T
authors.lastname: Mori
mesh_terms: D000375:Aging; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D018736:DNA Adducts / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D004855:Equilenin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D004857:Equilin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D004966:Estrogens, Conjugated (USP) / Q000008:administration & dosage; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C
chemical_list: D000911:Antibodies, Monoclonal; D018736:DNA Adducts; D004966:Estrogens, Conjugated (USP); D004857:Equilin; C065250:4-hydroxy-equilenin; C065248:4-hydroxy-equilin; D004855:Equilenin
references.citation: Biochemistry. 2007 Jan 9;46(1):182-91
references.pmid: 17198388
references.citation: Chem Res Toxicol. 2002 Mar;15(3):305-11
references.pmid: 11896676
references.citation: J Mass Spectrom. 2001 Mar;36(3):317-28
references.pmid: 11312524
references.citation: Chem Res Toxicol. 2005 Nov;18(11):1737-47
references.pmid: 16300383
references.citation: Chem Res Toxicol. 2001 Dec;14(12):1654-9
references.pmid: 11743748
references.citation: Science. 1964 Aug 14;145(3633):709-10
references.pmid: 14168277
references.citation: Carcinogenesis. 2003 May;24(5):911-7
references.pmid: 12771036
references.citation: Biochemistry. 2009 Aug 4;48(30):7098-109
references.pmid: 19527068
references.citation: Carcinogenesis. 2006 Feb;27(2):178-96
references.pmid: 16272169
references.citation: J Invest Dermatol. 2002 Nov;119(5):1177-82
references.pmid: 12445209
references.citation: J Mol Biol. 2007 Aug 31;371(5):1151-62
references.pmid: 17603077
references.citation: Chem Res Toxicol. 2009 Jun;22(6):1129-36
references.pmid: 19368368
references.citation: Photochem Photobiol. 1991 Aug;54(2):225-32
references.pmid: 1780359
references.citation: DNA Repair (Amst). 2004 Nov 2;3(11):1475-82
references.pmid: 15380103
references.citation: Biochemistry. 2006 May 16;45(19):6187-94
references.pmid: 16681391
references.citation: Chem Res Toxicol. 1998 Feb;11(2):94-101
references.pmid: 9511900
references.citation: Lancet. 2003 Aug 9;362(9382):419-27
references.pmid: 12927427
references.citation: Chem Res Toxicol. 2006 Mar;19(3):450-6
references.pmid: 16544951
references.citation: Cancer Res. 1995 Oct 1;55(19):4347-51
references.pmid: 7671246
references.citation: Chem Res Toxicol. 1998 Sep;11(9):1105-11
references.pmid: 9760286
references.citation: J Am Soc Mass Spectrom. 2003 May;14(5):482-91
references.pmid: 12745217
references.citation: Obstet Gynecol. 1995 Feb;85(2):304-13
references.pmid: 7824251
references.citation: Biochemistry. 2005 Nov 8;44(44):14565-76
references.pmid: 16262256
references.citation: Chem Res Toxicol. 2008 Sep;21(9):1739-48
references.pmid: 18680315
references.citation: Genes Cells. 1999 Nov;4(11):607-18
references.pmid: 10620008
references.citation: Proc Natl Acad Sci U S A. 1999 Oct 12;96(21):11922-7
references.pmid: 10518552
references.citation: Mutat Res. 1983 Aug;112(4):191-200
references.pmid: 6888408
references.citation: JAMA. 2002 Feb 13;287(6):734-41
references.pmid: 11851540
references.citation: N Engl J Med. 1997 Jun 19;336(25):1769-75
references.pmid: 9187066
references.citation: N Engl J Med. 1995 Jun 15;332(24):1589-93
references.pmid: 7753136
references.citation: Chem Res Toxicol. 2008 Jan;21(1):93-101
references.pmid: 18052105
references.citation: Chem Res Toxicol. 2008 May;21(5):1064-73
references.pmid: 18416538
references.citation: Carcinogenesis. 1984 Mar;5(3):373-7
references.pmid: 6423306
references.citation: Epidemiology. 1994 Jul;5(4):415-21
references.pmid: 7918811
references.citation: Chem Res Toxicol. 1998 Oct;11(10):1113-27
references.pmid: 9778307
references.citation: J Natl Cancer Inst. 1981 Sep;67(3):515-9
references.pmid: 6944523
references.citation: Chem Res Toxicol. 1999 Feb;12(2):204-13
references.pmid: 10027800
references.citation: Biochemistry. 2004 Sep 7;43(35):11312-20
references.pmid: 15366941
references.citation: Proc Natl Acad Sci U S A. 1981 Jul;78(7):4124-7
references.pmid: 6794029
references.citation: JAMA. 2002 Jul 17;288(3):334-41
references.pmid: 12117398
references.citation: JAMA. 2009 Jul 15;302(3):298-305
references.pmid: 19602689
references.citation: Chem Res Toxicol. 2003 Jun;16(6):695-707
references.pmid: 12807352