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 lacI gene using the polymerase STOP assay, following reaction in vitro with α- acetoxytamoxifen and horseradish peroxidase (HRP)/H₂O₂ activated 4- hydroxytamoxifen. For both compounds, most adduct formation occurred on guanines. However, one adenine, within a run of guanines, generated a strong polymerase STOP site with activated 4-hydroxytamoxifen, and a weaker STOP site with α-acetoxytamoxifen at the same location. In Escherichia coli the lac I gene reacted with 4-hydroxytamoxifen was more likely to be mutated (2 orders of magnitude) than when reacted with α-acetoxytamoxifen, despite the greater DNA adduct formation by α-acetoxytamoxifen. This correlates with the greater predicted ability of activated 4-hydroxytamoxifen adducts to disrupt DNA structure than α-acetoxytamoxifen adducts. For lac I reacted with activated 4-hydroxytamoxifen, a hot spot of base mutation was located in the region of the only adenosine adduct. No mutational hot spots were observed with α-acetoxytamoxifen. Our data clearly shows a lack of correlation between gross adduct number, as assayed by ³²P-postlabeling and mutagenic potential. These data indicate the importance of minor adduct formation in mutagenic potential and further that conclusions regarding the mutagenicity of a chemical may not be reliably derived from the gross determination of adduct formation.