Neuronal Nitric Oxide Synthase-Mediated ...

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Sep 10, 2015 - IgG were obtained from BD Biosciences (Germany). Assessment of Cell Viability by Muse Cell Analyzer. HT22 cells were seeded onto 6-well ...
Neuronal Nitric Oxide Synthase-Mediated Genotoxicity of 2-Methoxyestradiol in Hippocampal HT22 Cell Line Magdalena Gorska, Michal A. Zmijewski, Alicja Kuban-Jankowska, Maciej Wnuk, Iwona Rzeszutek & Michal Wozniak Molecular Neurobiology ISSN 0893-7648 Mol Neurobiol DOI 10.1007/s12035-015-9434-5

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Author's personal copy Mol Neurobiol DOI 10.1007/s12035-015-9434-5

Neuronal Nitric Oxide Synthase-Mediated Genotoxicity of 2-Methoxyestradiol in Hippocampal HT22 Cell Line Magdalena Gorska 1 & Michal A. Zmijewski 2 & Alicja Kuban-Jankowska 1 & Maciej Wnuk 3 & Iwona Rzeszutek 3 & Michal Wozniak 1

Received: 9 April 2015 / Accepted: 10 September 2015 # Springer Science+Business Media New York 2015

Abstract 2-methoxyestradiol, metabolite of 17β-estradiol, is considered a potential anticancer agent, currently investigated in several clinical trials. This natural compound was found to be effective towards great number of cancers, including colon, breast, lung, and osteosarcoma and has been reported to be relatively non-toxic towards non-malignant cells. The aim of the study was to determine the potential neurotoxicity and genotoxicity of 2-methoxyestradiol at physiological and pharmacological relevant concentrations in hippocampal HT22 cell line. Herein, we determined influence of 2methoxyestradiol on proliferation, inhibition of cell cycle, induction of apoptosis, and DNA damage in the HT22 cells. The study was performed using imaging cytometry and comet assay techniques. Herein, we demonstrated that 2methoxyestradiol, at pharmacologically and also physiologically relevant concentrations, increases nuclear localization of neuronal nitric oxide synthase. It potentially results in DNA strand breaks and increases in genomic instability in hippocampal HT22 cell line. Thus, we are postulating that naturally occurring 2-methoxyestradiol may be considered a physiological modulator of neuron survival. Keywords 2-methoxyestradiol . Neuronal nitric oxide synthase . Reactive nitrogen species . DNA damage . Hippocampus * Magdalena Gorska [email protected] 1

Department of Medical Chemistry, Medical University of Gdansk, Gdansk 80-211, Debinki 1 St, Poland

2

Department of Histology, Medical University of Gdansk, Gdansk, Poland

3

Department of Genetics, University of Rzeszow, Rzeszow, Poland

Abbreviations C Control COMT Catechol-O-methyltransferase DDR DNA damage response DSBs DNA double-strand breaks E2 17β-estradiol 2-ME 2-methoxyestradiol RNS Reactive nitrogen species SSBs DNA single-strand breaks

Introduction The hippocampus is a brain structure responsible for memory formation, retrieval, learning, and emotional behavior. Impairment in neurogenesis, or synaptic contact in brain structures, may result in numerous human brain pathologies and neurodegenerations like Alzheimer’s (AD), Parkinson’s (PD), or Huntington’s diseases [1]. Moreover, direct relationship between memory impairment and functional changes in the hippocampus has been reported. The importance of the hippocampus in the pathophysiology of depressive disorders has also been evidenced [2–4]. Hippocampal volume has been reduced in patients with depressive disorders [4, 5]. Circulating hormones have turned out to be very effective tools to analyze brain structure and function. Estrogens, a type of female sex steroids, are very important factors in the brain and may also play an important role in non-reproductive functions [6]. Interestingly, they are able to directly impact on other behaviors in brain regions, thus controlling mood and cognition, enhancing memory, or inducing neuroprotection [6]. Brain estrogens may be derived from peripheral steroidogenic organs via the blood stream, and from de novo synthesis at specific brain regions from steroid precursors or cholesterol

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[7]. Neurosteroids are synthetized from cholesterol in the central and peripheral nervous system through mechanisms independent of peripheral steroidogenic organs [7]. The hippocampus is one of the regions that may actively synthetize 17β-estradiol (E2) as a neurosteroid [6, 8, 9]. Interestingly, in adult rats, the level of E2 in the hippocampus was six times higher than in plasma, indicating synthesis de novo [10]. The complex actions of estrogens in the hippocampus have emerged from extensive in vitro and in vivo studies. E2 results in enhancement of such Bhippocampal^ tasks as place learning, navigation, or object place learning in rodents [6]. 2methoxyestradiol (2-ME) is a physiological metabolite of E2. 2-ME is synthesized by hydroxylation and subsequent O-methylation catalyzed by catechol-O-methyltransferase (COMT) [11, 12]. COMT is widely distributed in the hippocampus where it catabolizes the catecholamine neurotransmitters and influences cognitive function, regulates dorsal hippocampal neurochemistry, and modulates hippocampusdependent behaviors [13, 14]. Thus, 2-ME may be a metabolite of E2 also in brain structures. Serum concentrations of 2ME range from 0.03 nM in men to as much as over 0.030 μM in pregnant women [15–18]. Though, neuroprotective effects of E2 are well known, there are inconsistent data concerning action of 2-ME [19–22]. Previously, we determined that pharmacologically relevant concentrations of 2-ME (1 and 10 μM) induce apoptosis in HT22 cells [19]. The possible neurotoxic effects of 2-ME have also been stated by Picazo and coworkers [20]. On the other side, 2-ME has been reported to provide neuroprotection [21]. It also reduced apoptosis after stroke due to inhibition of hypoxia-inducible factor Hif1α [21, 22]. Herein, we investigated the plausible genotoxic and neurotoxic effects of 2-ME used at physiologically and pharmacologically relevant concentrations in hippocampal HT22 cells.

Materials and Methods Cell Line and Culture Conditions Immortalized mouse hippocampal HT22 cell line was kindly gifted by Kelvin Davies from the University of Southern California (USC; Los Angeles, CA, USA). The cells were cultured at 37 °C in a humidified atmosphere saturated with 5 % CO2 using Dulbecco’s modified Eagle’s medium supplemented with 10 % heat-inactivated fetal bovine serum and a penicillin (100 mg/mL)/streptomycin (100 mg/mL) cocktail (Sigma-Aldrich). Cell Treatment Hippocampal HT22 cells were treated with various concentrations of 2-ME and time depending on design of experiments.

In order to exclude potential influence of serum-derived hormones, all experiments were performed in the medium supplemented with charcoal-stripped FBS (Sigma-Aldrich, Poland). Reagents Tissue culture media, antibiotic cocktail, fetal bovine sera, 2methoxyestradiol, and preNω-nitro-l-arginine methyl ester (LNAME) were purchased form Sigma-Aldrich (Poland). Antirabbit IgG were purchased from Abcam (Cambridge, UK). Mouse antibodies against neuronal nitric oxide synthase (nNOS) and secondary polyclonal rat antibody against mouse IgG were obtained from BD Biosciences (Germany). Assessment of Cell Viability by Muse Cell Analyzer HT22 cells were seeded onto 6-well plates at a density of 300000 cells/well. After 24 h of culturing in the standard medium, the cells were treated with 2-ME for additional 48 h. The cells were then pelleted and incubated with Muse Count and Viability Reagent according to manufacturer’s protocol (Merck, Poland). Afterward, the cells (5000 events/sample) were analyzed, and the fluorescent signals of were detected using the Muse Cell Analyzer (Merck, Poland). The cell viability assay contains a mix of two DNA-intercalating fluorescent dyes that clearly identify all nucleated cells, live and dead (Merck, Poland). The results were then analyzed by Muse 1.4 analysis software. Each experiment was performed at least three times. Cytokinesis-Block Micronucleus Assay To evaluate 2-ME-induced micronuclei generation in HT22 cells, after 24-h treatment with 2-ME, a cytokinesis-block micronucleus (CBMN) assay was performed using BD™ Gentest Micronucleus Assay Kit using the standard protocol according to the manufacturer’s instructions. Shortly, a total of 500 binucleated cells per well [23, 24] were scored using an In Cell Analyzer 2000 (GE Healthcare, UK) equipped with a high-performance CCD camera. For a positive control, 24-h treatment with 100 ng/mL mitomycin C was used. nNOS Immunostaining For nNOS immunostaining, interphase nuclei were used. After treatment with 2-ME in the 96-well plate, HT22 cells were fixed with 3.7 % formaldehyde containing 0.1 % Triton X-100 in phosphate-buffered saline (PBS) for 20 min. Subsequently, the cells were incubated with 1 % bovine serum albumin (BSA) in phosphate-buffered saline containing 0.25 % Triton X-100 (PBST) at room temperature for

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30 min. After washing with PBST, the cells were incubated with a mouse monoclonal antibody against nNOS (diluted 1:50 in PBST containing 1 % BSA (PBST-BSA); BD Biosciences, Germany) overnight at 4 °C, and with a Fourier transform infrared (FITC)-conjugated, secondary polyclonal rat antibody against mouse IgG (diluted 1:1000 in PBSTBSA; BD Biosciences, Germany) at room temperature for 1 h. Nuclei were visualized with Hoechst 33342. Digital cell images were captured with an In Cell Analyzer 2000 (GE Healthcare, UK) equipped with a high-performance CCD camera. To analyze cellular nNOS content and localization, In Cell Analyzer software (In Cell Analyzer Investigator) was used. The fluorescence density is presented in relative fluorescence units (RFUs). As a positive control, treatment with 0.1 mg/mL nocodazole was used. 53BP1 Immunostaining For 53BP1 immunostaining, interphase nuclei were used. After treatment with 2-ME (0.01, 0.1, 1, and 10 μM) in the 96-well plate, HT22 cells were fixed with 3.7 % formaldehyde containing 0.1 % Triton X-100 in PBS for 20 min. Subsequently, the cells were incubated with 1 % bovine serum albumin (BSA) in PBST at room temperature for 30 min. After washing with PBST, the cells were incubated with a rabbit polyclonal antibody against 53BP1 (diluted 1:200 in PBST-BSA (PBST containing 1 % BSA); Novus Biologicals, Poland) overnight at 4 °C, and with, a FITC-conjugated, secondary polyclonal antibody against rabbit IgG (diluted 1:200 in PBST-BSA; BD Biosciences, Germany) at room temperature for 1 h. Nuclei were visualized with Hoechst 33342. Digital cell images were captured with an In Cell Analyzer 2000 (GE Healthcare, UK) equipped with a high-performance CCD camera. 53BP1 foci were scored per nucleus. Nitric oxide (NO) Level Imaging of reactive nitrogen species (RNS) production in living cells based on fluorescent indicators has been reported as a fast, sensitive and selective method [25]. After treatment with 2-ME (0.01, 0.1, and 1 μM), nitric oxide (NO) levels were evaluated using fluorogenic probe DAF-FM DA (5 μM) and 10-min incubation time in PBS buffer. NO-specific fluorescent signals were captured with an In Cell Analyzer 2000 (GE Healthcare, UK) equipped with a high-performance CCD camera. For NO-positive control, a NO donor—MAHMA NONOate (1 mM) was used. Comet Assay The analysis was performed as previously described [26]. DNA double-strand breaks (DSBs) and DNA single-strand

breaks (SSBs) were assessed with neutral and alkaline single-cell microgel electrophoresis (comet assay), respectively. After treatment with 2-ME (0.01, 0.1, and 1 μM), HT22 cells were suspended in PBS, mixed with low melting agarose (0.7 %), added to agarose slides, lyzed with proteinase K (0.5 mg/mL), and reduced glutathione (2 mg/ml) in lysis solution (1.25 M NaCl, 50 mM EDTA, 100 mM Tris–HCl, 0.01 % N-lauroylsarcosine sodium salt, pH 10) at 37 °C for 2 h and proceeded with electrophoresis (neutral comet assay buffer, 100 mM Tris–HCl, 0.5 M NaCl, 1 mM EDTA, 0.2 % DMSO, pH 10, and alkaline comet assay buffer: 1 mM EDTA, 0.2 % DMSO, 300 mM NaOH, pH >12). The slides were then stained with 0.25 μM YOYO-1 (Invitrogen Corporation, Grand Island, NY, USA) in 2.5 % DMSO and 0.5 % sucrose mounted with a coverslip and digital comet images were immediately captured with an Olympus BX61 fluorescence microscope equipped with a DP72 CCD camera and Olympus CellF software. The CCD capture conditions were: exposure time 81 ms, magnification 400·×. At least 100 comets were measured per each sample triplicate using AutoComet Software http://autocomet.com/index.php (TriTek Corp). The Tail Moment - tail length multiplied by the fraction of total DNA in the tail (Tail moment=tail length × % of DNA in the tail) was scored as general parameter to DNA integrity assessment.

Cell Cycle Analysis After treatment with 2-ME, HT22 cells were stained with a mixture of Hoechst 33342 (2.5 μg/ml) and Cell Trace™ Calcein Red-Orange AM (2.5 μM) (Life Technologies, Poland) in a serum-free Dulbecco’s modified Eagle’s medium (DMEM) medium at 37 °C for 30 min, washed with PBS, and subjected to cell cycle analysis using an In Cell Analyzer 2000 (GE Healthcare, UK) equipped with a high-performance CCD camera. The cell cycle of all the cells was established using the ImageJ software and DNA Cell Cycle Plug-In (MBF Collection).

Statistical Analysis The results represent the mean ± SD from at least three independent experiments. All microscopic evaluations were done on randomized and coded slides. Differences between control samples versus 2ME-treated samples were assessed with one-way analysis of variance (ANOVA) with post hoc testing using a Dunnett’s multiple comparison test. A p value of less than 0.01 was considered to correspond with statistical significance. Data were analyzed using GraphPad Prism (GraphPad Software, Inc., version 6, USA).

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Results Effects of Physiological and Pharmacological Relevant Concentrations of 2-ME on HT22 Cell Viability Previously, we demonstrated that 2-ME at high pharmacologically relevant concentrations inhibited cell growth and induced cell death in hippocampal HT22 and osteosarcoma 143B cell lines [19]. Herein, the hippocampal HT22 cells were treated with different concentrations of 2-ME for 48 h: physiological (0.01 nM–0.01 μM) and pharmacological (0.1 μM, 1 μM), and cell viability was analyzed. The concentrations range used in the study was based on the literature data [15–20]. We observed that treatment with physiological relevant concentrations does not significantly affect the HT22 cell viability. Only treatment with pharmacological relevant concentrations of 2-ME equaled to 0.1 and 1 μM increased the number of dead cells by 5 %±1.5, 10.1 %±1.1 as compared with the control (C), respectively (Fig. 1a).

with 0.01, 0.1, and 1 μM 2-ME as compared with the control (8.9 %), respectively (Fig. 1b). We have also observed inhibition of cell cycle in subG1 phase what indicates induction of apoptosis. The cells in subG1 phase were increased up to 1.3, 3.4, and 19.5 % after 24 h incubation with 0.01, 0.1, and 1 μM 2-ME as compared with the control (0 %), respectively (Fig. 1b). The cells in S phase were decreased to 25.6, 16.1, and 13.2 % after 24 h incubation with 0.01, 0.1, and 1 μM 2ME as compared with the control (32.2 %), respectively (Fig. 1b). The obtained data are consistent with those from HT22 cell viability.

2-ME Increases Level of Cytosolic and Nuclear Nitric Oxide Synthase nNOS

Next, we determined an impact of 2-ME on an inhibition of the cell cycle in HT22 cells. We selected representative concentrations of 2-ME: physiologically (0.01 μM) and pharmacologically (0.1 and 1 μM) relevant. Cell cycle analysis was determined by imaging cytometry using staining with a mixture of Hoechst 33342 and Cell Trace™ Calcein Red-Orange. As demonstrated, 24 h treatment with all used concentrations of 2-ME increased the number of HT22 cells in G2 phase of the cell cycle (Fig. 1b). The percent of HT22 cells in G2 phase was equaled to 17.1, 29.9, and 27.4 % after 24 h incubation

Previously, we demonstrated that pharmacologically relevant concentrations of 2-ME resulted in increased level of total nNOS leading to hippocampal cell death [19]. Herein, we determined time-dependent level and localization of nNOS isoforms (cytosolic, nuclear) after stimulation with 2-ME at physiological and pharmacological relevant concentrations (Fig. 2). The hippocampal HT22 cells were treated with physiologically (0.01 μM) and pharmacologically (0.1 μM, 1 μM) relevant concentrations for 0.5–24 h. After selected time points, the localization of the nNOS isoform was determined by using imaging cytometry and specific anti-nNOS antibodies. As demonstrated in Fig. 2a, b, the total level of nNOS was significantly increased by 17.2, 10, and 17.9 % after incubation with 0.01 μM 2-ME for 0.5, 1, and 24 h as compared with the control, respectively. The treatment with both pharmacological concentrations of 2-ME resulted in increase of the total nNOS level after 0.5 and 24 h of incubation; 0.1 μM 2-ME increased the level of total nNOS by 16.2 % and 12 % after 0.5

Fig. 1 Impact of 2-ME on HT22 cells viability and inhibition of cell cycle. a 2-ME decreases viability of HT22 cells only after treatment with pharmacologically relevant concentrations (0.1 and 1 μM). HT22 cells were treated with different concentrations of 2-ME (0.01 nM–1 μM) for 48 h. The cell viability was then analyzed by Muse Cell Analyzer. b Cell cycle analysis in HT22 cells treated with 2-ME. After 24-h treatment with

2-ME (0.01, 0.1, and 1 μM), the cell cycle arrest was determined using In Cell Analyzer 2000. The cells were stained with a mixture of Hoechst 33342 and Cell Trace™ calcein red-orange. Values are the mean±SE of three independent experiments (N=6 replicate cultures). The absence of error bar denotes a line thickness greater than the error. *P< 0.01; **P