Carcinogenesis Advance Access originally published online on May 19, 2005
Carcinogenesis 2005 26(10):1811-1820; doi:10.1093/carcin/bgi132
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Carcinogenesis vol.26 no.10 © Oxford University Press 2005; all rights reserved.
Novel genotoxicity assays identify norethindrone to activate p53 and phosphorylate H2AX
Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center, The Sidney Kimmel Comprehensive Cancer Center and 1 Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA
* To whom correspondence should be addressed at: Cancer Research Building 464, Department of Oncology, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD 21231, USA. Tel: +1 410 614 3314; Fax: +1 443 287 4653; Email: sk{at}jhmi.edu
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 
-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.
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