Novel genotoxicity assays identify norethindrone to activate p53 and phosphorylate H2AX

Eike Gallmeier, Jordan M. Winter, Steven C. Cunningham, Saeed R. Kahn 1 and Scott E. Kern *

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 {gamma}-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.

Abbreviations: {gamma}-H2AX, phosphorylated H2AX; CHO, Chinese hamster ovary; DMSO, dimethyl sulfoxide; DSBs, double-strand breaks; ICH, International Conference on Harmonization; PBS, phosphate-buffered saline; SBE, Smad-binding element; TBS, Tris-buffered saline; UDS, unscheduled DNA-synthesis


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There are more than 25 million organic and inorganic substances currently registered in the Chemical Abstracts Service registry, and ~4000 new substances are added each day (www.cas.org). Even when only a small fraction of these substances are intended for environmental release or exposure to humans, there is an increasing need for simple and rapid screening of substances and particularly pharmaceuticals for their genotoxic potential. Additionally, some of the previously introduced chemicals, tested decades ago with the methods available at that time, should be considered for re-testing with more reliable genotoxicity screening assays. There is an abundance of different tests used for genotoxicity screening in vitro and in vivo. As these are neither highly specific nor highly sensitive, a battery of complementary tests is currently required prior to the release of a chemical.

The International Conference on Harmonization (ICH) developed guidelines for the genotoxicity testing of pharmaceuticals and recommends a standard battery of three assays (1): an assay for gene mutation in bacteria, a chromosomal aberrations assay in mammalian cells in vitro (or a mouse lymphoma assay in vitro assessing mutations at the thymidine kinase locus) and a chromosomal aberrations assay in vivo using rodent hematopoietic cells. Drugs that are negative in all the three tests are considered to provide a sufficient level of safety. Additional testing is recommended for compounds yielding equivocal results or being negative but having shown effects in carcinogenicity bioassays in the absence of the evidence for a non-genotoxic mechanism. This includes the measurement of DNA adducts, DNA-repair/recombination and DNA-strand breaks (1).

Although the above battery detects significant heritable genetic changes (i.e. point mutations, chromosomal deletions, translocations, whole chromosome loss and mitotic recombination) (2), these genetic endpoints represent irreversible, fixed changes and therefore late events in a cell. Specific assays for the detection of earlier events, i.e. primarily non-mutational lesions, including DNA adducts, sister-chromatid exchanges, unscheduled DNA-synthesis (UDS) and DNA single-strand breaks or especially double-strand breaks (DSBs), are not routinely performed in genotoxicity screening. Small amounts of DSBs are usually repaired, i.e. that they are reversible lesions. Failed repair of these lesions, however, can lead to mutations, chromosomal aberrations and cell transformation (3). Furthermore, drugs inducing low amounts of DNA DSBs, which are applied repeatedly over extended time periods, represent a constant threat to genomic integrity. This long-term, low mutation rate might be an important carcinogenic risk. Therefore, there is a need for rapid and highly sensitive assays that assess the earlier DNA lesions, especially DSBs, to complement conventional genotoxicity screening.

Assays commonly used to assess DSBs include the comet assay, alkaline elution and pulsed-field gel electrophoresis. Because of their direct physical assessment of DNA damage, they are highly specific but lack sensitivity. Novel assays measuring a cell's response to DNA damage gain considerable sensitivity due to signal amplification, but can lack specificity due to the indirect assessment of DNA damage. These latter assays could provide valuable surrogate markers for DNA damage when combined with specific assays, but are not yet employed for genotoxicity screening.

In this study, we propose a technical algorithm to screen for DNA damage with special attention to DSBs. As a fast, sensitive and high-throughput primary screen, we applied the p53-reporter assay, testing 16320 compounds (DIVERSet, ChemBridge, San Diego, CA) and 700 drugs (Prestwick library, Prestwick Chemical, Washington, DC). These results were published (4). Assessment of p53 activation as a surrogate marker for DNA damage was suggested as a genotoxicity screening tool in the past (57), offering sensitivity and some specificity for DNA strand breaks (8). Because several non-genotoxic mechanisms exist for p53 activation (9), this assay is best followed by a confirmatory method. H2AX, a variant of histone H2A, is rapidly phosphorylated on Ser-139 in the vicinity of DSBs (10,11). Therefore, it represents a highly specific cellular response to DSBs. The sensitive detection of phosphorylated H2AX (termed {gamma}-H2AX) has very recently been proposed as a novel genotoxicity screen (12). We applied the immunofluorescent detection of {gamma}-H2AX as a validating test to complement our p53-reporter assay. Using this combination we found norethindrone, a common drug prescribed for contraception and hormone replacement therapy, to be probably genotoxic and, in extrapolation, potentially mutagenic and carcinogenic.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents and chemicals
Norethindrone was obtained from two suppliers (Sigma Chemical Co., St Louis, MO and Prestwick Chemical). Four lots from Sigma were used with indistinguishable results. Norethindrone was initially dissolved in dimethyl sulphoxide (DMSO) (Sigma) at 25 or 10 mg/ml. Therefore, the highest tested concentrations of 100 µg/ml norethindrone contained 0.4 or 1.0% DMSO, respectively. The p53-reporter and Smad-binding element (SBE) reporter cell lines developed by us have been described previously (4,13). We chose the colorectal cancer cell line RKO for the p53-reporter assays because it is a robust, fast-growing and naturally immortal p53-wild-type cell line that is easy to handle in tissue culture and reporter assays.

Cytotoxicity assay
RKO colorectal cancer cells were cultured in white-walled 96-well tissue-culture microplates and treated with norethindrone at 0.1, 1, 10 and 100 µg/ml for 4 and 24 h. Appropriate dilutions of DMSO were used as negative controls. Dead cells were removed by phosphate-buffered saline (PBS) wash. After cell lyses in 100 µl H2O for 1 h, 100 µl of 0.5% Picogreen (Molecular Probes, Eugene, OR) was added per well. Fluorescence was measured using a fluorometer (Fusion, Universal Microplate Analyzer; Perkin Elmer, Shelton, CT). Relative cellular DNA was calculated with the DMSO-treated samples defined as representing 100% survival. Nine independent experiments were performed, with each data point of a given experiment reflecting at least triplicate wells.

p53- and SBE-reporter assays
Cells were treated for 24 h after plating at ~70% confluence in white-walled 96-well tissue-culture microplates. Norethindrone was used at concentrations of 0.1, 1, 10 or 100 µg/ml for 1, 2, 4 and 24 h. Trichostatin A at 0.3 µg/ml was used as a positive control for reporter expression (14) and DMSO as a negative control. After the addition of Steady-Glo luciferase substrate (Promega, Madison, WI), light emission was measured using a photodetector (Trilux, Wallac, Gaithersburg, MD). Relative light emission was calculated with the emission of the corresponding DMSO-treated samples defined as having a value of 1. Three independent experiments were performed, with each data point of a given experiment reflecting at least triplicate wells.

Immunoblotting
Immunoblotting was performed using standard protocols. After norethindrone treatment, cells were scraped into the lysis buffer (50 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 0.1% bromphenol blue and 10% ß-mercaptoethanol). Proteins were denatured at 95°C for 5 min, separated by SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking in Tris-buffered saline/0.3% Tween (TBS–T)/5% milk, the membranes were incubated overnight with either a monoclonal mouse anti-human phospho-H2AX antibody (1:1000; Upstate, Waltham, MA) or a monoclonal mouse anti-human p53 antibody (Ab-2; Calbiochem, San Diego, CA), or monoclonal mouse anti-human caspase-3/caspase-9 antibodies (1:1000; Upstate). Membranes were washed and incubated with a secondary goat anti-mouse antibody (1:10000; Pierce, Rockford, IL) for 1 h 30 min. Detection was performed using SuperSignal Pico reagents (Pierce) according to the manufacturer's instructions. Equal protein loading was verified by membrane staining of the total protein with Fast Green (USB, Cleveland, OH).

Immunofluorescence
Cells were grown on coverslips in six-well tissue-culture microplates and treated at ~70% confluency with norethindrone at concentrations of 0.1, 1, 3, 10, 30 and 100 µg/ml for 1, 2, 4 and 24 h. DMSO was used as a negative control. Treated cells were fixed for 15 min in PBS/4% paraformaldehyde, washed, and fixed for 1 min in methanol at –20°C. After permeabilization for 10 min in TBS/0.5% Triton X-100 and blocking for 30 min in TBS/2% bovine serum albumin/0.5% Triton X-100, cells were incubated with a primary monoclonal mouse anti-human {gamma}-H2AX antibody (1:200; Upstate) for 2 h. Subsequently, the cells were washed and incubated with an Alexa 488 goat anti-mouse IgG secondary antibody (Molecular Probes) for 1.5 h. After washing, nuclei were counterstained with Hoechst 33258 (Sigma) at 10 µg/ml. Slides were mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL) and analyzed using a fluorescent microscope (Zeiss Axiovert 135, Maple Grove, MN) and the Metamorph 4.6 software (Universal Imaging, Downingtown, PA). Pictures were acquired using a Nikon DXM1200 digital camera (Nikon, Melville, NY). Exposure time and software settings were kept constant for all the samples within each experiment.

Cell cycle analysis
Cells were exposed to norethindrone at 10 µg/ml for 4 and 24 h, washed in PBS and fixed in PBS/3.7% formaldehyde/0.5% NP40 (US Biochemical, Cleveland, OH). Cells were incubated with Hoechst 33258 (10 µg/ml). DNA content was analyzed by flow cytometry (LSR I; Becton Dickinson Franklin Lakes, NJ, USA). Per sample, 10000 events were acquired. The data were processed using the Cell Quest software (Becton Dickinson).

Neutral comet assay
RKO and Chinese hamster ovary (CHO) cells were treated with norethindrone at 10 µg/ml for 4 h. Cells irradiated with 20 Gy and harvested immediately were used as a positive control and DMSO as a negative control. Cells were detached using 0.05% trypsin, diluted to 500000 cells/ml, mixed with pre-warmed 0.75% low melting agarose (1:10, v/v) and immediately layered onto pre-treated slides (Trevigen, Gaithersburg, MD). Gels were incubated at 4°C for 30 min to adhere to the slides. Cells were then lysed in pre-chilled lysis buffer (2.5 M sodium chloride, 100 mM EDTA, pH 10, 10 mM Tris, 1% sodium lauryl sarcosinate and 1% Triton X-100) for 30 min. After a wash step in neutral TBE buffer (0.9 M Tris, 0.9 M boric acid and 25 mM disodium EDTA), electrophoresis was performed in TBE at 1 V/cm for 15 min. Slides were then washed, submerged in 70% ethanol for 5 min and air-dried overnight. The gels were stained with SYBR Green (Trevigen) and imaged using a fluorescent microscope with a 10x objective and a Nikon Digital Eclipse DXM 1200 camera. CometScore software (TriTek, Sumerduck, VA) was used to morphometrically integrate the mean tail moment of 50 randomly selected comets from each gel. At least three independent experiments were averaged for each data point.

Chromosomal aberrations assay
Cells were grown in 75 cm2 flasks and treated at ~70% confluency with norethindrone at 10 and 100 µg/ml for 24 h. DMSO was used as a negative control. Prior to harvest, colcemid (Invitrogen, Carlsbad, CA) was added at a final concentration of 0.01 µg/ml. After 4 h, the cells were trypsinized, pelleted and resuspended in pre-warmed hypotonic solution. After incubation at 37°C for 30 min, cells were pelleted and resuspended in fresh fixative (3:1 methanol/acetic acid). The fixative was changed three times prior to slide preparation. Slides were made according to the standard cytogenetic methods and stained with Leishman. For each sample, 50 metaphases were analyzed for breakage and other structural abnormalities by our institution's cytogenetics core facility. Owing to the screening nature of the experimental design, no formal statistical procedures were applied. For data interpretation, we applied the criteria of Sofuni et al. (15,16), which were based on background chromosomal aberration rates and originally applied to CHL cells. The frequency of cells with aberrations in untreated CHL cells was ≤4% and therefore comparable with the frequency of cells with aberrations in untreated CHO cells (≤3%) (15) and untreated RKO cells (≤2%, our unpublished data, Gallmeier,E. and Kern,S.E.). The criteria were defined as follows. The test was considered negative when the fraction of aberrant cells was <5%, inconclusive, when >5% but <10%, and positive, when 10% or more. Results between 10 and 20% were considered weakly positive.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Assessment of norethindrone purity
To minimize the risk of the contaminating components, we used norethindrone from two independent suppliers (Sigma and Prestwick), using four different lots from Sigma. We re-tested norethindrone in the p53-reporter assay over a period of several years. The structure of norethindrone was confirmed by nuclear magnetic resonance mass spectrometry. No impurities or contaminants were detected (data not shown).

Cytotoxicity of norethindrone
At the time of the genotoxicity assays, no cell death was observed microscopically after treatment with norethindrone at concentrations up to 10 µg/ml in any cell line. Some amount of cell death was observed at 100 µg/ml. To quantify the cytotoxic effects of norethindrone, we further measured DNA content representing the total cell numbers in our samples. RKO cells were treated with norethindrone at concentrations 0.1, 1, 10 or 100 µg/ml, or the respective DMSO concentrations for 4 and 24 h at ~70% confluency, which represents the confluency used in the genotoxicity assays. Cytotoxicity was estimated using the DNA ratio (cell numbers) of DMSO-treated samples versus norethindrone-treated samples. No differences in survival were observed at any norethindrone concentration after the treatment for 4 h. After 24 h, the relative survival of norethindrone-treated samples was >80% in all experiments, with no detectable cytotoxicity at 0.1 and 1 µg/ml and an increasing cytotoxicity from 10 µg/ml (range 83–100% survival) to 100 µg/ml (82–100% survival). The cytotoxicity of norethindrone was dependent upon cell confluency at the time of the treatment. When cells were treated at lower confluencies than used in our genotoxicity assays (20–50% instead of 70%), we observed a greater effect on relative cell survival, decreasing to 83% at 1 µg/ml (range 83–100% survival), 60% at 10 µg/ml (60–78%) and 36% at 100 µg/ml (36–69%).

p53-reporter activation upon norethindrone treatment
RKO cells containing a luciferase-based reporter of p53 activity (p53R cells) were used as a primary screen for the p53 activation of 16320 compounds (DIVERSet, ChemBridge) and 700 drugs (Prestwick) using a screening dose of 2 µg/ml (4). Norethindrone induced a borderline activation of p53 (2-fold) at 2 µg/ml (data not shown) that led us to further investigate the genotoxic potential of this drug.

p53R cells were treated with norethindrone at 0.1, 1, 10 and 100 µg/ml for 1, 2, 4 and 24 h. DMSO alone at respective concentrations did not induce p53 and was therefore subsequently used as a negative control. We observed a 4-fold p53-reporter activation 24 h after the treatment with norethindrone at 10 µg/ml, which increased to 5-fold at 100 µg/ml. No p53-reporter activation was observed at earlier time points (Figure 1). In comparison, etoposide induced a 2-fold p53-reporter activation at the screening dose of 2 µg/ml and a maximal activation of 18-fold at 60 µg/ml. Camptothecin induced a 3-fold p53-reporter activation at 2 µg/ml and a maximal activation of 4-fold at 30 µg/ml (4).



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Fig. 1. p53-reporter activation after norethindrone treatment. RKO cells were treated with 0.1, 1, 10 or 100 µg/ml norethindrone and assessed in the p53-reporter assay after 1, 2, 4 and 24 h. Relative light emission was calculated, defining the light emission of DMSO-treated samples as ‘1’. Data were averaged from three independent experiments. Error bars represent SEM.

 
To exclude the non-specific enhancement of gene transcription (17), we additionally tested norethindrone in Panc-1 cells containing a luciferase-based SBE reporter (13). No SBE reporter activation was observed 24 h after the norethindrone treatment at 10 µg/ml (relative light emission of norethindrone treated samples = 0.97/SEM of three experiments = 0.03), a dose that induced a 4-fold increase in the p53-reporter assay.

We did not find p53 activation upon treatment with any other structurally or functionally related drug contained in the Prestwick library, including androsterone, betamethasone, epiandrosterone, estradiol, estradiol-17ß, hydrocortisone, lynestrenol, metanephrine, methylprednisolone, norethynodrel, norgestrel, progesterone and tamoxifen.

p53 protein upregulation and H2AX phosphorylation upon norethindrone treatment
To confirm p53 upregulation at the protein level and to screen for H2AX phosphorylation upon norethindrone treatment, we performed immunoblotting using antibodies that recognize p53 and {gamma}-H2AX, respectively. p53 and {gamma}-H2AX were both detected at low levels in the untreated control samples and were markedly upregulated 4 h after the treatment with norethindrone at 10 µg/ml (Figure 2).



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Fig. 2. p53 and {gamma}-H2AX protein upregulation after norethindrone treatment. RKO cells were treated with 10 µg/ml norethindrone or 0.1% DMSO for 4 h and assessed for p53 and {gamma}-H2AX protein upregulation using western blotting. The membrane was probed with an antibody against p53 (upper panel) and {gamma}-H2AX (middle panel), respectively. Equal protein loading was confirmed by membrane staining for total protein (lower panel). Experiments were performed at least in triplicate and representative results are shown.

 
Immunofluorescent detection of {gamma}-H2AX
We used immunofluorescence to verify the immunoblotting results at the single cell level and to examine the intranuclear distribution and focus formation of {gamma}-H2AX. DMSO at the concentrations used in the norethindrone experiments did not increase {gamma}-H2AX as compared with untreated cells and was subsequently used as a negative control. For comparability, exposure time and software settings for picture acquisition were kept constant for all samples within each experiment (Figure 3, each panel representing individual experiments). Norethindrone induced {gamma}-H2AX became visible at 10 µg/ml and increased in a dose-dependent manner when assessed at 30 and 100 µg/ml (Figure 3A, upper panel). At 10 µg/ml, H2AX phosphorylation was detectable as soon as 1 h after the treatment and did not significantly decrease after 4 or 24 h (Figure 3A, lower panel). This is consistent with a recent report showing sustained {gamma}-H2AX accumulation at 24 h after irradiation, when most DSBs were already repaired (18). Treatment of cells with etoposide at 50 or 250 µg/ml, or irradiation at 5, 10 or 20 Gy caused a more pronounced induction of {gamma}-H2AX than the treatment with norethindrone. Also, the pattern of {gamma}-H2AX localization upon norethindrone treatment differed strikingly from that upon irradiation or etoposide. Upon irradiation, an increase in nuclear {gamma}-H2AX was observed in all cells, located in small and discrete foci that were homogeneously distributed throughout the nucleus (Figure 3B, lower panel). Upon etoposide, very bright {gamma}-H2AX fluorescence of the whole nucleus, without discernible foci, was observed in all cells at 50 µg/ml (Figure 3B, upper panel) and this increased in intensity at 250 µg/ml (data not shown). However, when decreased exposure time and software editing (scaling) were applied, distinct and homogeneously distributed foci were revealed, similar to the pattern seen upon irradiation (data not shown). In contrast, norethindrone at a concentration of 10 µg/ml increased {gamma}-H2AX in most but not all cells, producing fewer and larger foci than irradiation or etoposide (Figure 3B, both panels). Use of decreased exposure time, software editing, higher magnification and nuclear counterstaining revealed those large foci to be accumulations of smaller foci, which were accentuated around nucleolar structures (Figure 3C). We performed blocking experiments using a 10-, 100- or 10000-fold molar excess of norethindrone, as compared with the primary antibody, during the incubation with the fixed cells. {gamma}-H2AX immunolocalization remained unchanged, providing no evidence for an artifact attributable to norethindrone-antibody binding.



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Fig. 3. {gamma}-H2AX immunofluorescence after norethindrone treatment. RKO cells were treated with the indicated concentrations of the indicated drugs for the indicated time periods, fixed and stained with an antibody against {gamma}-H2AX (green). Nuclei were counterstained with Hoechst 33258 (blue). (A) Dose (upper panel) and time (lower panel) kinetics. (B) Effects of potent DSB-inducers (irradiation and etoposide) on H2AX phosphorylation as compared with the effect of norethindrone. (C) A higher magnification was used to elicit the pattern of norethindrone-induced H2AX phosphorylation at a higher resolution. Arrows mark nucleolar structures. (A–C) Representative images from independent experiments.

 
The above immunofluorescence experiments in RKO cells were extended to a total of four cells lines, two of malignant (RKO and PL-5) and two of non-malignant (HEK293 and CHO) origin, to exclude a cell-line-specific phenomenon. Although the background levels of H2AX phosphorylation in the untreated cells differed markedly between the cell lines, being higher in HEK293 and CHO than in RKO and PL-5, a significant upregulation of {gamma}-H2AX was reproducibly observed in all cell lines (data not shown).

In addition we tested whether H2AX phosphorylation would occur upon treatment of cells with structurally and functionally related drugs (cyproterone acetate, ethynodiol diacetate, megestrol acetate, norgestrel and progesterone) or other hormones (estradiol, hydrocortisone, metanephrine and tamoxifen). These drugs were tested at concentrations of 10 and 100 µg/ml. We found progesterone to be strongly cytotoxic at 100 µg/ml and megestrol to cause an increase in apoptosis at 100 µg/ml with a concomitant increase of immunodetectable {gamma}-H2AX in the apoptotic cells, but we did not find induction of {gamma}-H2AX in non-apoptotic cells upon treatment with any of these drugs (data not shown).

Assessment of apoptosis upon norethindrone treatment
As apoptotic cells have intense H2AX phosphorylation (19), we excluded apoptosis as a possible cause for the observed {gamma}-H2AX upregulation. In the {gamma}-H2AX immunofluoresence assay, apoptotic cells could be easily distinguished from non-apoptotic cells by their characteristic nuclear morphology along with intense {gamma}-H2AX-staining. We observed no increase either in sporadic apoptotic cells or in {gamma}-H2AX-marked nuclei having microscopically apoptotic characteristics in our experiments (4/500 apoptotic cells 24 h after treatment with 0.1% DMSO as compared with 3/500 apoptotic cells 24 h after treatment with norethindrone at 10 µg/ml in a representative experiment).

Additionally, we performed immunoblotting with antibodies against caspase-3 and caspase-9. Untreated samples (RKO) displayed barely detectable levels of cleaved (activated) caspase-3 or caspase-9. These levels did not increase 24 or 48 h after norethindrone treatment at 10, 30 and 100 µg/ml (data not shown).

Assessment of chromosomal aberrations upon norethindrone treatment
We performed cytogenetic studies to determine the clastogenic potential of norethindrone. RKO and CHO cells were treated with norethindrone at 10 and 100 µg/ml for 24 h. Subsequently, 50 metaphases of each sample were analyzed for breakage and other structural abnormalities (Table I). According to the criteria of Sofuni and colleagues (15,16), our assay was negative for RKO cells, but weakly positive for CHO cells.


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Table I. Chromosome aberrations after norethindrone treatment

 
Cell cycle profile of norethindrone-treated cells
To test whether the DNA-damage induced by norethindrone is accompanied by a detectable cell cycle arrest, we performed flow cytometric cell cycle analysis after the treatment with norethindrone. Norethindrone at 10 µg/ml did not change the cell cycle distribution at 4 h (data not shown) or 24 h after the treatment (Figure 4).



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Fig. 4. Cell cycle analysis after norethindrone treatment. RKO cells were treated with 10 µg/ml norethindrone or left untreated. Cells were fixed 24 h after treatment, stained with Hoechst 33258 and analyzed by flow cytometry. Percentages of cells in G1-, and S- and G2/M-phase of the cell cycle are indicated. No arrest was detected.

 
Neutral comet assay for assessment of DSBs in norethindrone-treated cells
We employed the neutral comet assay, which should detect DSBs induced by norethindrone if present in moderate quantities. In either of the two cell lines (RKO and CHO), we did not see an increase in mean tail moment 4 h after treatment with norethindrone at 1, 10 or 100 µg/ml as compared with DMSO-treated control cells. In contrast, the positive controls (irradiation at 20 Gy and hydrogen peroxide at 100 µM) had significant increases in mean tail moment (Figure 5 and data not shown). Because the neutral comet assay is more specific for detecting DSBs, in contrast to single-strand breaks and alkali-labile sites, but less sensitive than the alkaline version of the comet assay (20,21), we additionally applied the alkaline comet assay to detect any damage induced by norethindrone. We did not observe an increase in mean tail moment in the alkaline comet assay (data not shown).



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Fig. 5. Neutral comet assay after norethindrone treatment. The neutral comet assay was performed on RKO and CHO cells treated with 0.1% DMSO (4 h), 20 Gy irradiation (30 min) or 10 µg/ml norethindrone (4 h). In each experiment, 50 cells were analyzed per sample and the mean tail moment was calculated using CometScore. (A) Representative pictures of one experiment. (B) Averaged data from 2–5 experiments. Error bars represent SEM.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we found that norethindrone activated p53 specifically and in a dose-dependent manner in human cells in vitro. Norethindrone also induced the phosphorylation of H2AX in various cell lines of non-malignant and malignant origin. These two findings are known as sensitive indicators of the creation of DSBs. Potential impurities or contaminants that might have occurred during the manufacture of norethindrone were not identified. Therefore, our data strongly suggest that—at least in vitro—norethindrone is capable of inducing DNA DSBs. The specificity of our finding is further corroborated by the fact that a panel of structually and/or functionally related drugs gave negative results, without exception, in both the assays.

The DNA damage appeared to be rapid after the exposure to norethindrone. p53 protein upregulation occurred as early as 4 h after the treatment, confirming that p53 activation followed a similar time course as H2AX phosphorylation. The p53-reporter assay peaked at 24 h, as expected for immediate p53 induction, owing to the longer half-life of luciferase.

Norethindrone-induced phosphorylation of H2AX differed quantitatively, as well as qualitatively, from the effects of known DSB-inducers. Quantitatively, norethindrone caused less of an increase in {gamma}-H2AX than did etoposide at 50 µg/ml or irradiation at 5, 10 and 20 Gy. Qualitatively, norethindrone caused a different intranuclear distribution of {gamma}-H2AX. Foci were distributed homogenously throughout the nucleus upon irradiation or etoposide, whereas foci were particularly accentuated around nucleolar structures upon norethindrone treatment.

Owing to the rapid repair of low levels of DNA damage, an asynchronous cell population generally does not arrest long enough to be detectable by automated cell cycle analysis; we failed to detect a cell cycle arrest upon norethindrone treatment, supporting the view that norethindrone causes only low levels of damage. We were also not able to detect DSBs upon norethindrone treatment by the neutral comet assay. This particular assay might be the most sensitive of the conventional assays having specificity for DSBs (22); therefore, norethindrone would be expected to be negative in other, less sensitive assays for DSBs (e.g. alkaline elution, pulsed-field electrophoresis). As norethindrone was negative even in the more sensitive (but less specific) alkaline comet assay (20,21), the other assays were not performed.

To detect potential clastogenic effects of norethindrone, we applied an assay similar to the chromosome aberrations assay of the ICH three-test battery. As extensive research has already been done in the past on norethindrone using standard chromosome aberration assays, which yielded both positive (23,24) and negative results (25,26), we focused on concentrations that had clearly induced p53 activation and H2AX phosphorylation in our experience and therefore employed two higher doses (10 and 100 µg/ml) in two cell lines. We obtained equivocal results, RKO being negative and CHO weakly positive. However, these results should be considered preliminary, because a standard chromosome aberrations assay for genotoxicity screening calls for the testing of at least three different concentrations of the drug (16), and a formal statistical analysis of the results would require many more replicates.

Norethindrone is one of the most commonly used drugs for female contraception and hormone replacement therapy (27), but its genotoxic potential is not settled. The International Agency for Research on Cancer classifies norethindrone to be ‘possibly carcinogenic to humans’ (Group 2B), based mainly on ‘limited evidence for carcinogenicity to animals’ (28). Several studies using various methods were performed to determine the genotoxic potential of norethindrone [reviewed in (29) and (30)]. These studies produced variable and somewhat inconsistent results. As concerns the standard three-test battery recommended by the ICH, one must consider the following reports: norethindrone is non-mutagenic in the Ames assay (23,31); norethindrone does (23) or does not (25,26) induce chromosomal aberrations in cultured human lymphocytes and does increase non-classical chromosomal aberrations in mouse bone marrow cells (24); norethindrone does (23) or does not (24) significantly increase micronuclei formation in mouse bone marrow cells and does increase micronclei formation in mouse lymphocytes (23), but is inconclusive in rat liver (32). Norethindrone is also positive in the liver foci assay (32), but does not cause DNA adducts in rat liver (33). Norethindrone does (23) or does not (26) increase the amount of sister-chromatid exchanges in mouse and human cells. Finally, norethindrone increases unscheduled DNA synthesis dose-dependently in male rat hepatocytes (34,35), but inconclusively in human hepatocytes (35).

Rodent carcinogenicity assays show norethindrone to increase the incidence of both benign and malignant tumors: norethindrone increases the occurrence of benign liver tumors in male mice (28) and in male (28) and female rats (36), benign mammary tumors in male (28) and female rats (36), uterine polyps in female rats (37), pituitary tumors in female mice (28) and male rats (36), granulosa-cell tumors in the ovaries of female mice (28), neoplastic nodules in the liver of rats (37), malignant mammary tumors in male and female rats (36) and in combination with ethynylestradiol, hepatocellular carcinoma in female rats (38,39).

In summary of the literature, there is as yet no clear consensus on the carcinogenicity of norethindrone. According to the ICH guidelines (equivocal results in standard genotoxicity testing), additional experiments would be recommended to further clarify the genotoxic potential of norethindrone. Our study suggests that norethindrone induces DSBs, both complementing and adding a new aspect to the prior literature. Also, the previously reported negative results in the Ames assay (as the gold standard for mutagenicity screening) are consistent with our findings, for DSB-inducing agents are systematically missed in this assay.

A prior report appears to support the theory of direct DNA damage induced by norethindrone. Blakey and White (34) found an increase in UDS after norethindrone treatment at 15 µg/ml. This phenomenon was linked to the presence of a 17{alpha}-ethynyl substituent, because only the steroids with this 17{alpha}-ethynyl substituent had an effect on UDS in their study (5/6 drugs compared with 0/4 drugs). Therefore, we tested steroids with and without the 17{alpha}-ethynyl substituent in our assays. In the {gamma}-H2AX assay, drugs with a 17{alpha}-ethynyl substituent included norethindrone, ethynodiol diacetate and norgestrel. Drugs without a 17{alpha}-ethynyl substituent included progesterone, cyproterone and megestrol acetate. In the p53-reporter assay, drugs with a 17{alpha}-ethynyl substituent included norethindrone, norgestrel, lynestrenol and norethynodrel. Drugs without a 17{alpha}-ethynyl substituent included progesterone, estradiol, estradiol-17ß, androsterone and epiandrosterone. Except for norethindrone, none of these drugs induced either p53 accumulation or H2AX phosphorylation. Therefore, our data do not support the above hypothesis linking the 17{alpha}-ethynyl substituent to the capability of steroids to bind DNA and to induce DNA damage (34). The question as to whether norethindrone interacts with DNA and thus directly mediates DNA damage, remains to be addressed in future studies.

There are other caveats about our study, some of which apply to the interpretation of genotoxicity screening results in general. Genotoxicity testing at high doses, up to the insoluble range, is required by the ICH (1). Although a few of the above-cited studies described the lowest effective dose (LED) of norethindrone to be as low or even lower than the LED in our study [induction of sister-chromatid exchanges and chromosomal aberrations at 1 µg/ml (23), increased UDS at 1.5 µg/ml (35) or 15 µg/ml (34)], most of the studies had employed higher doses, up to the millimolar range. Nevertheless, the effects of norethindrone in our study, observed at low micromolar concentrations, need to be considered in the light of the therapeutic blood levels of norethindrone, which are in the lower nanomolar range (35,40), i.e. at least, a 100-fold lower. Therefore, the physiological relevance of our studies, with respect to the typical pharmacological doses of norethindrone, is uncertain. A number of concentration-dependent artifacts, for example, should be considered.

First, drug precipitation can occasionally cause DSBs via a yet unknown mechanism (41). We indeed detected precipitation at 100 µg/ml norethindrone by light microscopical analysis. However, we did not detect precipitation at concentrations of 10 µg/ml or lower, a dose at which norethindrone produced both a p53 response and an increase in {gamma}-H2AX. Therefore, the observed results in our assays cannot be explained by precipitation.

Second, some non-genotoxic drugs induce DSBs exclusively under highly cytotoxic conditions (42), causing a particular dilemma in the interpretation of such results: the formation of DSBs at the highly cytotoxic doses of a drug might reflect secondary effects in dying cells rather than primary genotoxic effects, and yet, genotoxic effects observed only at high experimental concentrations can be a clue to physiologic long-term genotoxic risk. An arbitrary general consensus defines a reduction of >50% in cell growth at the time of sampling as a suitable upper limit of cytotoxicity for routine genotoxicity screening (41). We did not observe a severe reduction in cell viability at the timepoint at which norethindrone clearly induced H2AX phosphorylation (4 h after treatment). Furthermore, no reduction in cell survival >20% was observed at the timepoints when p53 activation was most apparent (up to 24 h after treatment), even for the highest dose used (100 µg/ml). When the cells were plated at lower confluencies than used for the p53-reporter and {gamma}-H2AX assays, we did observe a stronger reduction in cell growth, up to 64%, after 24 h at the highest norethindrone dose. Thus, while we cannot definitely exclude that our H2AX-phosphorylation assays were complicated in part by cytotoxicity, it seems unlikely that cytotoxicity alone accounted for the observed effects. Moreover, the p53-reporter assay is a robust readout for p53 activation even at high cytotoxicity levels, and indeed, positive results in this assay are known to often be masked rather than augmented artificially under highly cytotoxic conditions (4).

Third, apoptosis causes intense H2AX phosphorylation (19). Upon microscopical scoring of nuclei in the immunofluorescence experiments, we observed no increase in apoptotic cells upon treatment with norethindrone. Similarly, using immunoblotting, we found no evidence for norethindrone-induced cleavage of caspase-3 or caspase-9. Therefore, apoptosis did not account for the observed H2AX phosphorylation induced by norethindrone in our experiments.

Our data indicate that norethindrone exhibits genotoxic properties in the experimental setting, but their relevance in regard to human exposure still remains as a question. Given the oral dosing of 0.1–5 mg/day, some organs (stomach, bowel and liver) would be exposed to much higher concentrations than are reflected in the low nanomolar peripheral serum values. Certain tissues would be exposed to norethindrone repeatedly over extended time periods. In general, mutagenic/carcinogenic effects of drugs at long-term/low-dose ingestion cannot be correctly simulated in short-term/high-dose experiments and cannot be definitely proven or disproven by these means. Therefore, the implications of long-term effects of norethindrone with respect to its ability to induce DSBs in certain human tissues cannot be predicted. It seems reasonable, however, to consider that drugs with similar therapeutic profile and comparable side-effects, but which are negative in genotoxicity testing, should be clinically preferred over those that are positive.

In summary, we applied a combination of two novel assays for genotoxicity screening that measure DNA damage indirectly. These assays rely on cellular signal amplification, and are therefore more sensitive than conventional direct assays. This led to the identification of a drug that potentially induced small levels of DNA DSBs that would not have been detected by the comet assay, a conventional and particularly sensitive assay for direct DSB assessment. We suggest that our combination of assays could complement common genotoxicity screening tests with special regard to the sensitive detection of DNA DSBs. Furthermore, these assays could be applied for various purposes as a highly sensitive, stand-alone combination for the detection of DSBs.


    Acknowledgments
 
We would like to thank L.A.Morsberger of the cytogenetics core facility at Johns Hopkins for the cytogenetic analyses and J.R.Brody, E.S.Calhoun and J.D.Yager for the helpful discussions. This work was funded by NIH grant CA 62924 and CA 68228. E.G. was supported by the Deutsche Forschungsgemeinschaft (DFG GA 762/1-1).

Conflict of Interest Statement: None declared.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 17, 2005; revised May 12, 2005; accepted May 13, 2005.





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