2006 Fiscal Year Final Research Report Summary
The possible role of equine estrogen-derived DNA adducts in the increased risk of cancers on estrogen replacement therapy
| Project/Area Number |
17510048
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Risk sciences of radiation/Chemicals
|
|---|
| Research Institution | Nara Medical University |
|---|
Principal Investigator |
MORI Toshio Nara Medical University, School of Medicine, Associate professor, 医学部, 助教授 (10115280)
|
|---|
| Project Period (FY) |
2005 – 2006
|
| Keywords | estrogen / female hormone / DNA adduct / breast cancer / ovarian cancer / endometrical cancer / premarin / monoclonal antibody |
|---|
| Research Abstract |
To study the mechanisms of estrogen replacement therapy-associated increased risk of breast, ovarian, and endometrical cancers, a mouse monoclonal antibody (40HEN-1) against equine estrogen-derived DNA adducts was prepared and used for analysis of the DNA adducts. Calf thymus DNA was exposed to 4-hydroxyequilenin (4-OHEN) in a buffer. The 4-0HEN-modified-DNA containing approximately 5.2 4-OHEN-dC adducts/10^4 nucleotides and 1.2 4-OHEN-dA adducts/10^4 nucleotides was electrostatically conjugated with mBSA, emulsified in Freund's adjuvant, and then used as an immunogen. The immunogen was administered intraperitoneally 4 times to BALB/c mice to induce the monoclonal antibodies. Using enzyme-linked immunosorbent assay (ELISA), the antibody obtained showed strong binding to the 4-OHEN-modified DNA but had undetectable or minimal binding to undamaged DNA, UV-irradiated DNA or acetylaminofluorene-modified DNA. Competitive inhibition experiments revealed that 4OHEN-1 binds most strongly to the Pk-4 form (one of diastereoisomers) of 4-OHEN-dC adduct and 4-OHEN-dA adduct in oligonucleotide, although the Pk-3 form of them is also recognized with slightly less efficiency. ELISA with 4OHEN-1 showed the linear dose response curve between the amount of 4-OHEN-DNA adducts coated in a well of plate and the antibody-binding to the adducts with a detection limit of 〜5 adducts/10^8 nucleotides. Using this assay, 4-OHEN-DNA adducts were detected in mammary carcinoma cells (MCF-7) which had been treated with equilenin (60 μM) for 6 days. The level of 4-0HEN-DNA adducts was 1.5 adducts in 10^7 nucleotides. We are waiting for the blood sample from the patients with estrogen replacement therapy, since the work has been accepted by the ethics committee of our university.
|
