Tamoxifen-DNA Adduct Formation in Rat Liver Determined by Immunoassay and P-Postlabeling

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 a-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 6 0.11 (mean 6 SE, n 5 18) fmol of (E)-a-(N-deoxyguanosinyl)tamoxifen (TAM-dG) adduct in TAM-DNA by DELFIA. For TAMDNA modified to 4.8 adducts in 10 nucleotides, 50% inhibition was at 20.6 6 6.6 (mean 6 SE, n 5 8) fmol of TAM-dG in TAM-DNA by DELFIA and at 0.92 6 0.11 (mean 6 SE, n 5 10) fmol of TAM-dG in TAM-DNA by CIA. No inhibition was observed in either assay with up to 20 mg (62.5 nmol of nucleotides) of unmodified DNA. The individual adducts TAM-dG and (Z)-a-(N-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 P-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 P-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 nucleotides (0.2 fmol of adducts per 20 mg of DNA).