Detection of tamoxifen-DNA adducts on lacI genes using DNA polymerase stop assay

Tamoxifen forms DNA adducts in rat liver and causes an increased mutation frequency at the lacI genes in the livers of lambda/lacI transgenic rats. Although an elevated occurrence of endometrial cancer is found in a small proportion of breast cancer patients treated with tamoxifen, there is conflicting evidence on whether or not low levels of DNA adducts are formed in humans.

Based on the finding that the progression of DNA/RNA polymerases on templates might be blocked by bulky DNA adducts, we successfully developed and used a polymerase stop assay to map the sites of adduct formation in the target lacI gene following its reaction in vitro with alpha-acetoxytamoxifen and horseradish peroxidase/H2O2 (HRP/H2O2) activated 4-hydroxytamoxifen.

Using a T4 DNA polymerase stop assay, adduct formation in the lacI gene of the plasmid constructs, after the reaction in vitro with alpha-acetoxytamoxifen and HRP/H2O2 activated 4-hydroxytamoxifen, was found to mainly occur with guanines. In particular, one site of adenosine adduction was found on a triplet of adenosines located between two runs of guanines.

The success of our development of DNA polymerase stop assay to map the sites of tamoxifen-DNA adducts formation will be very useful for the investigation of the mutagenicity/carcinogenicity of tamoxifen. The mutagenic potential of the tamoxifen adducted bases shall be further examined by transfecting the adducted plasmids into suitable human cell lines. Also, further investigations of the sequence specificity in specific oncogenes and tumor suppressor genes may be useful to explore the relationship between the occurrence of human endometrial cancer and tamoxifen treatment.