Quantitative detection of 4-hydroxyequilenin-DNA adducts in mammalian cells using an immunoassay with a novel monoclonal antibody.
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.
Okahashi
Yumiko
Y
Radioisotope Research Center, Department of Neurology and Medical Genetics Research Center, Nara Medical University School of Medicine, Kashihara, Nara 634-8521, Japan.
Iwamoto
Takaaki
T
Suzuki
Naomi
N
Shibutani
Shinya
S
Sugiura
Shigeki
S
Itoh
Shinji
S
Nishiwaki
Tomohisa
T
Ueno
Satoshi
S
Mori
Toshio
T
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
D000911:Antibodies, Monoclonal; D018736:DNA Adducts; D004966:Estrogens, Conjugated (USP); D004857:Equilin; C065250:4-hydroxy-equilenin; C065248:4-hydroxy-equilin; D004855:Equilenin
Biochemistry. 2007 Jan 9;46(1):182-91
17198388
Chem Res Toxicol. 2002 Mar;15(3):305-11
11896676
J Mass Spectrom. 2001 Mar;36(3):317-28
11312524
Chem Res Toxicol. 2005 Nov;18(11):1737-47
16300383
Chem Res Toxicol. 2001 Dec;14(12):1654-9
11743748
Science. 1964 Aug 14;145(3633):709-10
14168277
Carcinogenesis. 2003 May;24(5):911-7
12771036
Biochemistry. 2009 Aug 4;48(30):7098-109
19527068
Carcinogenesis. 2006 Feb;27(2):178-96
16272169
J Invest Dermatol. 2002 Nov;119(5):1177-82
12445209
J Mol Biol. 2007 Aug 31;371(5):1151-62
17603077
Chem Res Toxicol. 2009 Jun;22(6):1129-36
19368368
Photochem Photobiol. 1991 Aug;54(2):225-32
1780359
DNA Repair (Amst). 2004 Nov 2;3(11):1475-82
15380103
Biochemistry. 2006 May 16;45(19):6187-94
16681391
Chem Res Toxicol. 1998 Feb;11(2):94-101
9511900
Lancet. 2003 Aug 9;362(9382):419-27
12927427
Chem Res Toxicol. 2006 Mar;19(3):450-6
16544951
Cancer Res. 1995 Oct 1;55(19):4347-51
7671246
Chem Res Toxicol. 1998 Sep;11(9):1105-11
9760286
J Am Soc Mass Spectrom. 2003 May;14(5):482-91
12745217
Obstet Gynecol. 1995 Feb;85(2):304-13
7824251
Biochemistry. 2005 Nov 8;44(44):14565-76
16262256
Chem Res Toxicol. 2008 Sep;21(9):1739-48
18680315
Genes Cells. 1999 Nov;4(11):607-18
10620008
Proc Natl Acad Sci U S A. 1999 Oct 12;96(21):11922-7
10518552
Mutat Res. 1983 Aug;112(4):191-200
6888408
JAMA. 2002 Feb 13;287(6):734-41
11851540
N Engl J Med. 1997 Jun 19;336(25):1769-75
9187066
N Engl J Med. 1995 Jun 15;332(24):1589-93
7753136
Chem Res Toxicol. 2008 Jan;21(1):93-101
18052105
Chem Res Toxicol. 2008 May;21(5):1064-73
18416538
Carcinogenesis. 1984 Mar;5(3):373-7
6423306
Epidemiology. 1994 Jul;5(4):415-21
7918811
Chem Res Toxicol. 1998 Oct;11(10):1113-27
9778307
J Natl Cancer Inst. 1981 Sep;67(3):515-9
6944523
Chem Res Toxicol. 1999 Feb;12(2):204-13
10027800
Biochemistry. 2004 Sep 7;43(35):11312-20
15366941
Proc Natl Acad Sci U S A. 1981 Jul;78(7):4124-7
6794029
JAMA. 2002 Jul 17;288(3):334-41
12117398
JAMA. 2009 Jul 15;302(3):298-305
19602689
Chem Res Toxicol. 2003 Jun;16(6):695-707
12807352