Novel genotoxicity assays identify norethindrone to activate p53 and phosphorylate H2AX

Norethindrone is a commonly used drug for contraception and hormone replacement therapy, whose carcinogenic potential is still controversial. We applied a novel and particularly sensitive method to screen for DNA damage with special attention to double-strand breaks (DSBs) and identified norethindrone to be likely genotoxic and therefore potentially mutagenic: A p53-reporter assay served as a first, high-throughput screening method and was followed by the immunofluorescent detection of phosphorylated H2AX as a sensitive assay for the presence of DSBs. Norethindrone at concentrations of 2-100 μg/ml activated p53 and phosphorylated H2AX specifically and in a dose-dependent manner. No p53 activation or H2AX phosphorylation was detected using a panel of structurally/functionally related drugs. The overall amount of DNA damage induced by norethindrone was low as compared with etoposide and ionizing radiation. Consistently, norethindrone treatment did not cause a cell cycle arrest. DSBs were not detected with the neutral comet assay, a less sensitive method for DSB assessment than H2AX phosphorylation. Our findings in the p53-reporter and γ-H2AX assays could not be ascribed to common DSB-causing artifacts in standard genotoxicity screening, including drug precipitation, high cytotoxicity levels and increased apoptosis. Therefore, our study suggests that norethindrone induces DSBs in our experimental setting, both complementing and adding a new aspect to the existing literature on the genotoxic potential of norethindrone. As the effective concentrations of norethindrone used in our assays were ∼100- to 1000-fold higher than therapeutical doses, the significance of these findings with regard to human exposure still remains to be determined.