Resolution of DNA adduct formation at the nucleotide level and correlation with site specific propensity to mutation
2014-12-15T10:42:01Z (GMT) by
Tamoxifen, a non steroidal antioestrogen (Z-trans-1-[4-(dimethylaminoethoxy)phenyl]-1,2-diphenyl-1-butene), is widely used in the treatment of breast cancer, and is undergoing clinical evaluation as a chemopreventative in women thought to be at high risk of developing the disease. Tamoxifen is hepatocarcinogenic in rats, forming large numbers of tamoxifen DNA adducts when dosed over a period of time, but is inactive in standard genotoxicity tests. In this project I determined in vitro and in vivo DNA adduct formation at the nucleotide level from tamoxifen and selected metabolites. Sites of tamoxifen DNA adduct formation were mapped using the T4 DNA polymerase arrest and single stranded ligation assays. Following the reaction of plasmid DNA in vitro with -acetoxytamoxifen or horseradish peroxidase/H2O2 activated 4-hydroxytamoxifen, DNA adduct formation occurred predominantly on guanine. Preliminary studies in vivo also showed sites of tamoxifen DNA adduct formation on guanine with minor adduct formation on adenine. Plasmids reacted in vitro with activated 4-hydroxytamoxifen, were mutated 2 orders of magnitude more frequently than were plasmids reacted with -acetoxytamoxifen in E. coli. This occurred despite -acetoxytamoxifen forming a greater number of DNA adducts. The mutational hot spot in the bacterial lacI gene occurred in the region of the only adenine adduct for plasmids treated with activated 4-hydroxytamoxifen. These data indicated a lack of correlation between gross adduct number and mutagenic potential. This has implications for the interpretation of gross DNA adduct data.