Tamoxifen-DNA adduct formation in rat liver determined by immunoassay and P-32-postlabeling

Tamoxifen (TAM), a nonsteroidal antiestrogen used as a chemotherapeutic and chemopreventive agent for breast cancer, induces liver tumors in rodents a nd covalent DNA adduct formation in hepatic DNA. Here, we report the develo pment and validation of highly sensitive and specific immunoassays for the determination of TAM-DNA adducts. Rabbits were immunized with calf thymus D NA, chemically modified with alpha-acetoxytamoxifen to 2.4 adducts per 100 nucleotides, and the resulting antisera were characterized by competitive d issociation-enhanced lanthanide fluoroimmunoassay (DELFIA) and chemilumines cence immunoassay (CIA). Compared with DELFIA, the CIA has a much lower bac kground and a 20-fold increase in sensitivity. For the immnnogen TAM-DNA, 5 0% inhibition was at 2.0 +/- 0.11 (mean +/- SE, n = 18) fmol of (E)-alpha-( N-2-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 in hibition was observed in either assay with up to 20 mu g (62.5 nmol of nucl eotides) of unmodified DNA. The individual adducts TAM-dG and (Z)-alpha-(N- 2-deoxyguanosinyl)tamoxifen and the individual compounds TAM and 4-OH-TAM g ave DELFIA 50% inhibitions at 828, 2229, 5440,and 8250 fmol, respectively. For assay validation, TAM-dG levels were determined by DELFIA, CIA, and P-3 2-postlabeling in TAM-DNA samples modified in vitro to different levels, an d comparable values were obtained in all three assays. Further validation w as obtained in vivo in rat liver. DNA adducts of TAM mere 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-32-postlabeling, although the CIA gave an under estimation 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 mu g of DNA).