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Tamoxifen-DNA adduct formation in rat liver determined by immunoassay and 32P-postlabeling
R L Divi1, M R Osborne, A Hewer
1National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
Abstract:
Tamoxifen (TAM), a nonsteroidal antiestrogen used as a chemotherapeutic and chemopreventive agent for breast cancer, induces liver tumors in rodents and covalent DNA adduct formation in hepatic DNA. Here, we report the development and validation of highly sensitive and specific immunoassays for the determination of TAM-DNA adducts. Rabbits were immunized with calf thymus DNA, chemically modified with alpha-acetoxytamoxifen to 2.4 adducts per 100 nucleotides, and the resulting antisera were characterized by competitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) and chemiluminescence immunoassay (CIA). Compared with DELFIA, the CIA has a much lower background and a 20-fold increase in sensitivity. For the immunogen TAM-DNA, 50% inhibition was at 2.0 +/- 0.11 (mean +/- SE, n = 18) fmol of (E)-alpha-(N2-deoxyguanosinyl)tamoxifen (TAM-dG) adduct in TAM-DNA by DELFIA. For TAM-DNA modified to 4.8 adducts in 10(6) nucleotides, 50% inhibition was at 20.6 +/- 6.6 (mean +/- SE, n = 8) fmol of TAM-dG in TAM-DNA by DELFIA and at 0.92 +/- 0.11 (mean +/- SE, n = 10) fmol of TAM-dG in TAM-DNA by CIA. No inhibition was observed in either assay with up to 20 microg (62.5 nmol of nucleotides) of unmodified DNA. The individual adducts TAM-dG and (Z)-alpha-(N2-deoxyguanosinyl)tamoxifen and the individual compounds TAM and 4-OH-TAM gave DELFIA 50% inhibitions at 828, 2229, 5440, and 8250 fmol, respectively. For assay validation, TAM-dG levels were determined by DELFIA, CIA, and 32P-postlabeling in TAM-DNA samples modified in vitro to different levels, and comparable values were obtained in all three assays. Further validation was obtained in vivo in rat liver. DNA adducts of TAM were measurable in rat liver 24 h after a single i.p. dose of 45 mg TAM/kg body weight and after daily p.o. dosing for 7 days with 5.0, 10.0, and 20.0 mg TAM/kg body weight. In addition, TAM-DNA adducts disappeared slowly over 21 days in rats on a control diet that were first given p.o. TAM at 45 mg/kg/day for 4 days. In the rat experiments, TAM-DNA adduct levels determined by CIA compared well with those determined by 32P-postlabeling, although the CIA gave an underestimation at the highest doses. For rat liver samples, the detection limit by CIA was 3 adducts per 10(9) nucleotides (0.2 fmol of adducts per 20 microg of DNA).
Insights
Tamoxifen (TAM) can form DNA adducts in the liver, potentially leading to tumors. This study developed highly sensitive immunoassays, specifically chemiluminescence immunoassay (CIA), to detect these TAM-DNA adducts in rodent models.
Area of Science:
- Biochemistry
- Toxicology
- Molecular Biology
Background:
- Tamoxifen (TAM) is a vital antiestrogen drug for breast cancer treatment and prevention.
- TAM is known to induce liver tumors in rodents and form covalent DNA adducts in hepatic DNA.
- Sensitive detection methods for TAM-DNA adducts are crucial for understanding its toxicological mechanisms.
Purpose of the Study:
- To develop and validate highly sensitive and specific immunoassays for determining Tamoxifen-DNA (TAM-DNA) adducts.
- To compare the performance of chemiluminescence immunoassay (CIA) with dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) and 32P-postlabeling methods.
- To investigate the presence and persistence of TAM-DNA adducts in vivo in rat liver.
Main Methods:
- Development of immunoassays by immunizing rabbits with TAM-modified DNA.
- Characterization of antisera using competitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) and chemiluminescence immunoassay (CIA).
- Validation of assays using in vitro TAM-DNA samples and in vivo studies in rat liver following various TAM dosing regimens.
Main Results:
- The developed CIA demonstrated significantly higher sensitivity and lower background compared to DELFIA.
- TAM-DNA adducts were successfully detected in rat liver 24 hours after a single dose and after repeated dosing.
- Adducts showed slow disappearance over 21 days in rats, with CIA results correlating well with 32P-postlabeling, despite slight underestimation at high doses.
Conclusions:
- Highly sensitive and specific immunoassays (CIA and DELFIA) for TAM-DNA adducts have been successfully developed and validated.
- These assays enable the detection of TAM-DNA adducts in vivo in rodent liver, providing insights into TAM's genotoxicity.
- The findings support the use of these immunoassays for further research into the mechanisms of TAM-induced hepatocarcinogenesis.