Site-specific tamoxifen-DNA adduct formation: Lack of correlation with mutational ability in Escherichia coli

We have mapped sites of tamoxifen adduct formation, in the loci gene using the polymerase STOP assay, following reaction in vitro with alpha-acetoxyta moxifen and horseradish peroxidase (HRP)/ H2O2 activated 4-hydroxytamoxifen For both compounds, most adduct formation occurred on guanines. However, o ne adenine, within a run of guanines, generated a strong polymerase STOP si te with activated 4-hydroxytamoxifen, and a weaker STOP site with alpha-ace toxytamoxifen at the same location. In Escherichia coil the lac I gene reac ted with 4-hydroxytamoxifen was more likely to be mutated (2 orders of magn itude) than when reacted with alpha-acetoxytamoxifen, despite the greater D NA adduct formation by a-acetoxytamoxifen. This correlates with the greater predicted ability of activated 4-hydroxytamoxifen adducts to disrupt DNA s tructure than alpha-acetoxytamoxifen adducts. For lac I reacted with activa ted 4-hydroxytamoxifen, a hot spot of base mutation was located in the regi on of the only adenosine adduct. No mutational hot spots were observed with alpha-acetoxytamoxifen, Our data clearly shows a lack of correlation betwe en gross adduct number, as assayed by P-32-postlabeling and mutagenic poten tial. These data indicate the importance of minor adduct formation in mutag enic potential and further that conclusions regarding the mutagenicity of a chemical may not be reliably derived from the gross determination of adduc t formation.