Genotoxic Properties of Cyclopentenone ... - ACS Publications

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Genotoxic Properties of Cyclopentenone Prostaglandins and the Onset of Glutathione Depletion Gergely Morten Solecki, Isabel Anna Maria Groh, Julia Kajzar, Carolin Haushofer, Anne Scherhag, Dieter Schrenk, and Melanie Esselen* Department of Chemistry, Division of Food Chemistry and Toxicology, Technische Universität Kaiserslautern, Erwin-Schrödinger-Str. 52, 67663 Kaiserslautern, Germany

ABSTRACT: Prostaglandins are endogenous mediators formed from arachidonic acid by cyclooxygenases and prostaglandin synthases during inflammatory processes. The five-membered ring can be dehydrated, and α,β-unsaturated cyclopentenone PGs (cyPGs) are generated. Recent studies have been focused on their potential pharmacological use against inflammation and cancer. However, little is known so far about possible adverse health effects of cyPGs. We addressed the question whether selected cyPGs at a concentration range of 0.1−10 μM exhibit mutagenic and genotoxic properties in the hamster lung fibroblast V79 cell line and whether these effects are accompanied by a depletion of intracellular glutathione (GSH). The cyPGs 15-deoxyΔ12,14-prostaglandin J2 (15dPGJ2) and prostaglandin A2 (PGA2) significantly induced DNA damage in V79 cells after 1 h of incubation. Furthermore, a more pronounced increase in formamidopyrimidine-DNA glycosylase (FPG) sensitive sites, indicative of oxidative DNA-damage, was observed. The findings on DNA-damaging properties were supported by our results that 15dPGJ2 acts as an aneugenic agent which induces the amount of kinetochore positive micronuclei associated with an increase of apoptosis. The strong potency of cyPGs to rapidly bind GSH measured in a chemical assay and to significantly reduce the GSH level after only 1 h of incubation may contribute to the observed oxidative DNA strand breaks, whereas directly induced oxidative stress via reactive oxygen species could be excluded. However, after an extended incubation time of 24 h no genotoxicity could be measured, this may contribute to the lack of mutagenicity in the hypoxanthine phosphorybosyltransferase (HPRT) assay. In conclusion, potential in vitro genotoxicity of cyPG and a strong impact on GSH homeostasis have been demonstrated, which may be involved in carcinogenesis mediated by chronic inflammation.

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INTRODUCTION

responsible for their strong potency to form Michael adducts, e.g., with GSH. The formation of covalent protein adducts is associated with a modulation in protein function, which has been described in depth in the literature.1 Transcription factors such as activator protein-1 (AP-1), nuclear factor κB (NfκB), or

Cyclopentenone prostaglandins (Chart 1) are reactive lipidic mediators that arise by nonenzymatic dehydration of certain prostaglandins (PGs). PGs belong to the family of eicosanoids, being derived from arachidonic acid liberated from membrane phospholipids by the action of phospholipase A2.1,2 cyPGs are characterized by the presence of the electrophilic α,βunsaturated carbonyl moiety in the cyclopentene ring © 2013 American Chemical Society

Received: October 29, 2012 Published: January 22, 2013 252

dx.doi.org/10.1021/tx300435p | Chem. Res. Toxicol. 2013, 26, 252−261

Chemical Research in Toxicology

Article

Chart 1. Chemical Structures of the Used cyPGs: (A) PGA2, (B) PGB2, and (C) 15dPGJ2a

a

*, location of electrophilic carbon atoms. (Karlsruhe, Germany). Kaiser’s glycerol gelatin and methylene blue were obtained from Merck (Darmstadt, Germany). Cyclopentenone prostaglandines were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Cell Culture. Male Chinese hamster V79 lung fibroblasts (Deutsche Sammlung für Mikroorganismen and Zellkultur, DSMZ, Braunschweig, Germany) were grown in Dulbecco’s modified Eagle’s medium (DMEM) low glucose (1 g/L) (PAA, Coelbe, Austria). Human colon carcinoma cells HT29 (DSMZ, Braunschweig, Germany) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (5 g/L) (Invitrogen, Life Technologies, Karlsruhe, Germany). Cell culture medium was supplemented with 10% fetal calf serum (FCS; PAA, Coelbe, Austria) and 1% penicillin/ streptomycin (Invitrogen Life Technologies, Karlsruhe, Germany). Cells were cultured at 37 °C in a water-saturated atmosphere containing 5% CO2 and were routinely tested for the absence of mycoplasm contamination. Compounds were dissolved in DMSO and added to the medium to yield a final DMSO concentration of 0.5% (v/ v). Lactate Dehydrogenase (LDH)-Leakage Assay. Cytotoxicity was determined by measuring the lactate dehydrogenase (LDH) activity in the cell medium using a commercial LDH cytotoxicity assay kit (Cayman Chemical, Ann Arbor, MI, USA). V79 cells (2 ×104) in 120 μL of serum containing medium were seeded on 96-well plates and allowed to grow for 24 h. Thereafter, cells were incubated with the test compounds (0.1, 1, 2.5, 5. 7.5, and 10 μM) under serum-free cell culture conditions to avoid protein binding for an additional 24 h. Sodium dodecyl sulfate (0.1% v/v) was included as a positive control. Subsequently, the 96 well plate was centrifuged for 5 min at 37 °C and 400g, and 100 μL of the supernatant was transferred to a second 96 well plate. To every plate, a standard LDH calibration line with the following LDH activities (0, 62.5, 125, 250, 500, and 1,000 μU) was included as duplicates. One hundred microliter LDH-reaction solutions were added, the plate was gently shaken for 30 min at 25 °C and measured immediately with a plate reader at λex = 490 nm. The effects on the membrane integrity were calculated as μU per mL. Determination of Doubling Time. V79 cells (4000/well) were seeded in 24 well plates. After 24 h, the culture medium was removed, and cells were washed with phosphate buffered saline and were incubated with the respective cyPGs under serum free conditions for 1 h. After that, the incubation medium was removed, and the cells were cultivated in serum-containing medium. This step was repeated after 72 h. Cell number was counted in quadruplicate over one week every day. Comet Assay. The comet assay was performed according to the method of Tice et al.24 V79 cells (1 × 106) were seeded into Petri dishes (⌀ = 6 cm, 5 mL medium containing 10% FCS) and allowed to grow for 24 h prior to treatment. Cells were treated for 1 h with the solvent control or the test compounds under serum-free conditions. As a positive control, the redox cycler menadione was included. Thereafter, aliquots (70,000 cells) were centrifuged (10 min, 425g). The resulting cell pellet was resuspended in 65 μL of low melting agarose and distributed onto an encoded frosted glass microscope slide, precoated with a layer of normal melting agarose. The slides were coverslipped and kept at 4 °C for 10 min to allow solidification of the agarose. After removing the cover glass, slides were immersed for 1

nuclear factor-E2-related factor 2 (Nrf-2), proteins involved in the regulation of the cellular redox status, such as thioredoxin, thioredoxin reductase, and cytoskeletal proteins, have been characterized as target proteins of cyPGs.3−10 With regard to the cell signaling responses to cyPGs, those to 15dPGJ2 are the best understood so far. 15dPGJ2 binds as an endogenous ligand to the peroxisome proliferator-activated receptor γ (PPARγ).11,12 Furthermore, the postulated anti-inflammatory effects are associated with a modulation of the NF-κB pathway via direct inhibition of IkB kinases.13 In addition, 15dPGJ2 has been demonstrated to induce apoptosis, to inhibit cell growth and differentiation and to induce the expression of antioxidant response element (ARE)-related genes by activating the Nrf-2 pathway.14−17 However, cyPGs are generated during inflammatory responses. Chronic inflammation, frequently combined with chronic infection, has been recognized in epidemiological and mechanistic studies to be a critical component for tumor formation and progression, being associated with carcinogenesis in one of every five cancer patients worldwide.18,19 Among the factors formed during inflammation, low-molecular weight mediators, such as cyPGs may contribute to these effects, particularly if they are genotoxic. 15dPGJ2 was found to induce cell proliferation of the colorectal cancer cell line HCA7.20 Millán et al. published that 15dPGJ2 during 7,12dimethylbenz[a]anthracene treatment significantly increases the rate of formation, size, and vascularization of papilloma.21 The aim of the present study was to clarify whether selected cyPGs (PGA2, PGB2, and 15dPGJ2) affect cellular targets such as the antioxidative protein GSH and/or DNA. GSH is the major endogenous thiol antioxidant, having an extensive role in the regulation of cellular redox balance as well as the detoxification of exogenous and endogenous substances such as xenobiotics, ionizing radiation, or several cytokines.22,23 Therefore, the total intracellular GSH level can be used for the assessment of the cellular redox status. Genotoxic properties of the respective cyPGs were investigated by single cell gel electrophoresis (comet assay), the HPRT assay, and the micronucleus assay in V79 cells. In addition, to detect oxidative DNA damage, a modified comet assay protocol with the DNA repair enzyme formamido-pyrimidine-DNA-glycosylase (FPG) was used. Furthermore, investigations about the mode of action of cyPG genotoxicity being aneugenic or clastogenic were included.

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EXPERIMENTAL PROCEDURES

Chemicals. Dimethyl sulfoxide (DMSO), glutathione, ethidium bromide, menadione, mitomycin C (MMC), nocodazole (NOC), and 6-thioguanine were obtained from Sigma−Aldrich (Steinheim, Germany) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) from TCI Europe (Zwijndrecht, Belgium). 4′,6-Diamidino-2-phenylindole-dihydrochloride (DAPI) was purchased from Carl Roth 253



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h at 4 °C in lysis solution (89 mL of lysis stock solution; 2.5 mM sodium chloride, 100 mM EDTA, 10 mM Tris, and 1% w/v Nlaurylsarcosin sodium salt; 1 mL of Triton-X-100 and 10 mL of DMSO). For the additional detection of oxidative DNA damage, slides were washed three times in enzyme buffer (40 mM HEPES at pH 8.0, 0.1 M potassium chloride, 0.5 mM EDTA, and 0.2 mg/mL bovine serum albumin), covered with 50 μL of either enzyme buffer or FPG enzyme, and incubated for 30 min at 37 °C. Subsequently, DNA was allowed to unwind (300 mM NaOH and 1 mM EDTA, pH 13.5, 20 min, 4 °C) followed by horizontal gel electrophoresis at 4 °C for 20 min (300 mA = const). Thereafter, the slides were washed three times with 0.4 M Tris-HCl, pH 7.5, and stained with ethidium bromide (40 μL per coverslip, 20 μg/mL). Fluorescence microscopy was performed with a Zeiss Axioskop 50/AC (λex = 546 ± 12 nm; λem > 590 nm). Slides were subjected to computer-aided image analysis (Comet Assay IV System, Perceptive Instruments, Suffolk, Great Britain), scoring 50 images per slide randomly picked. For each concentration of test compound, two slides were processed and analyzed independently. The results were parametrized with respect to tail intensity (fluorescence intensity of the DNA in the comet tail calculated as percentage of overall DNA fluorescence intensity in the respective cell). In parallel to the comet assay, viability of the cells was determined by trypan blue exclusion. Dichlorofluorescein (DCF) Assay. The DCF assay was performed using dark 96 well culture plates (Corning Life Sciences, Amsterdam, The Netherlands) seeded with HT29 cells at a density of 40,000 in 100 μL of serum-containing medium per well, which were previously allowed to grow for 48 h. Afterward, the cell culture medium was removed, and cells were washed with phosphate buffered saline and incubated with 100 μL of 50 μM 2′,7′- dichlorofluorescindiacetate solution (DCFH-DA) for 30 min at 37 °C. The dye supernatant was removed, and the cells were washed twice with phosphate buffered saline. HT29 cells were incubated with cyPGs (0.1−10 μM). Menadione was included in the testing as a positive control for the induction of ROS. In the presence of intracellular ROS, cellular esterases generated 2′,7′-dichlorofluorescin (DCFH) that was oxidized to fluorescent 2′,7′-dichlorofluorescein (DCF). The increase in fluorescence intensity was measured with a fluorometer at λex = 485 nm and λem = 535 nm over a period of 3 h. Micronuclei Test and Apoptosis Staining. Briefly, 105,000 V79 cells were seeded onto Petri dishes (⌀ 60 mm) and kept for 24 h in an incubator (37 °C, 5% CO2). Cell culture medium was removed; the cells were rinsed off with 5 mL of phosphate buffered saline (PBS) and treated for 1 h with the test substances or for 24 h with the positive control mitomycin C (MMC, 0.6 μM) or with UVB (150 J/m2) to induce apoptosis. After removing the test compounds by medium replacement, the cells were incubated with FCS-containing medium for another 20 h and washed with PBS. Cells were fixed with 3 mL of Carnoy’s fixation solution (methanol/acetic acid 3:1) per Petri dish at −20 °C for 1 h. For staining, fixation solution was removed, and cells were incubated for 10 min at −20 °C with a 0.1% DAPI solution (49.9% v/v PBS, 49.9% v/v methanol, 0.1% Triton-X-100), washed with ice-cold methanol, and air-dried. Petri dishes were coverslipped, and the dried cell layers were then covered with about 50 μL of melted Kaiser’s glycerol gelatin, and coverslips were fixed on top. Fluorescence microscopy was performed with a Zeiss Axioskop. Micronuclei and apoptotic cells were always derived from at least three independent sets of experiments and from the evaluation of 2000 individual cells per concentration (1000/Petri dish). CREST-Staining. V79 cells were seeded onto cover slides in quadriPERM (Sarstedt, Nümbrecht, Germany) dishes. After 24 h in culture, the medium was removed, and test compounds were added for 60 min, except the positive control nocodazole (NOC, 0.5 μM), which was incubated for 16 h. Subsequently, serum-containing medium was added. After 20 h, cells were fixed with ice-cold methanol (1 h, 4 °C), and the cell membranes were made permeable with acetone. After a blocking step with goat serum for 1 h, the cells were incubated with the primary anticentromere protein A IgG (CREST) antibody (Antibodies Incorporated, Davis, CA, USA) for 1 h at 37 °C. Thereafter, cells were washed, and a FITC-conjugated antibody against

human IgG (Fab-specific, Sigma-Aldrich, Munich, Germany) was used as secondary antibody. Cells were washed with Sörensen-phosphate buffer (pH 8) and stained with a DAPI/propidium iodide/antifade solution. 103 nuclei per cover slide were counted using a Zeiss Laser Axiovert 200 microscope (Carl Zeiss AG, Göttingen, Germany). Thiol Reactivity. The spontaneous GSH reactivity of the test compounds was measured in a phosphate buffered system (A/B buffer 1.5 mL of buffer A, 25 mM KH2PO4 and 6 mM Na2EDTA; and 8.5 mL of buffer B, 125 mM K2HPO4 and 6 mM Na2EDTA) containing equimolar concentrations of glutathione (GSH) and the respective test compound within a total volume of 1 mL. The level of remaining GSH in the sample aliquot was measured photometrically (λ = 412 nm) due to the reaction with Ellman’s reagent [6 mM 5,5′-dithio-bis(2nitrobenzoic acid) to 5-thio-2-nitrobenzoate]. Glutathione (GSH) Assay. The GSH assay was performed with slight modifications according to the method of Tietze.25 V79 cells (1 × 106 or 5 × 105) were seeded onto Petri dishes (Ø 6 cm) and allowed to grow for 24 h. Subsequently, cells were incubated for 1 h with the test compounds or 24 h with the synthetic amino acid L(−)-buthionine sulfoximine (BSO), an irreversible inhibitor of the γglutamyl-cysteine synthetase, as positive control Cells were collected, and the viability was determined by trypan blue exclusion. After several washing steps with cold phosphate buffer, cells were centrifuged at 180g for 10 min at 4 °C. The resulting cell pellet was resuspended in 1 mL of A/B buffer (15 mL of buffer A, 125 mM KH2PO4 and 6 mM Na2EDTA; and 85 mL of buffer B, 125 mM K2HPO4 and 6 mM Na2EDTA) and centrifuged at 425g for 10 min at 4 °C. Thereafter, 360 μL of A/B buffer was added to the resulting cell pellet, and 2 × 10 μL of this cell suspension was used for protein quantification, whereas the remaining cell suspension was mixed with 350 μL of 10% (w/v) sulfosalicylic acid for cell lysis. Subsequently, the suspension was centrifuged for 10 min at 4 °C to remove the protein precipitant. For quantitative determination of total GSH (tGSH) status, 10 μL of the supernatant were mixed with 190 μL of the tGSH mixture [164 μL of A/B buffer, 20 μL of 6 mM 5,5′-dithio-bis(2- nitrobenzoic acid), 4 μL of 20 mM NADPH, and 2 μL of GSH reductase (GR) solution (50 U/ mL)]. In this reaction, GSSG was reduced to GSH by glutathione reductase (GR) and nicotinamide adenine dinucleotide phosphate (NADPH). Finally, the tGSH level of the cell (reduced GSSG and initial GSH) was measured photometrically (λ = 412 nm) due to the reaction with Ellman’s reagent. The tGSH content was expressed as nmol tGSH/mg protein. Therefore, the cellular protein of the cell suspension in the GSH assay was quantified with the BCA assay according to the protocol of Sigma-Aldrich (Bicinchoninic Acid Protein Assay Kit). The principle of the BCA assay relies on the formation of a Cu2+‑protein complex under alkaline conditions, followed by reduction of the Cu2+ to Cu+. The amount of reduction is proportional to the protein present. Absorbance was measured photometrically (λ = 592 nm). V79 Hypoxanthine Phosphoribosyltransferase (HPRT) Assay. The HPRT assay was performed according to the method previously reported by our group.26 V79 cells (1 ×106) were seeded onto 75 cm2 flasks in 10 mL of FCS-containing medium (10%) and kept for 24 h under standard conditions in an incubator. Then, cell culture medium was removed, and the flasks were rinsed with 5 mL of PBS. The test substances dissolved in DMSO were diluted with FCSfree medium, and 10 mL of this solution was added to each flask. The positive control was treated with 20 μM MNNG. After 1 h, supernatants were removed, and 10 mL of FCS-containing medium was added to the cells. After 24 h, cell culture medium was removed, the cell layer was rinsed with 5 mL of PBS buffer, and 1 mL of trypsin solution was added to each flask. V79 cells were suspended in 10 mL of FCS-containing medium and counted, and 1 × 106 V79 cells were transferred with 15 mL of FCS-containing medium to 75 cm2 flasks. After 48 h, cells were handled as described in the step before. Again, 1 × 106 cells were transferred into 75 cm2 flasks as described above and incubated for another 48 h. Cells were again trypsin-treated, and after that, 1 × 106 cells were transferred into 15 mL of thioguaninecontaining medium (500 mL of DMEM low glucose, 25 mL of FCS, 5 mL of Pen/Strep, 5 mL of 100 mM sodium pyruvate solution, 0.5 mL 254



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of 54 mM 6-thioguanine solution), each to three 75 cm2 flasks. As a vitality control (plating efficiency; PE), 240 cells each were suspended in 10 mL of FCS-containing medium and transferred to two Petri dishes (⌀ = 10 cm). After nine days, Petri dishes and incubation flasks were rinsed with 0.9% saline, and 5 mL of ice-cold ethanol was added to each vessel and kept for at least 15 min at −20 °C. After removal of ethanol and addition of 3 mL of methylene blue solution (0.5% in ethanol), the vessels were kept for at least 30 min at −20 °C. Then, the methylene blue solution was removed. V79 cells were carefully washed with tap water and air-dried. The colonies per vessel were counted, and the mean (number) was calculated for each treatment. The mutation frequency MF was calculated as MF = meanflask × 240/ meanplate.

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RESULTS Cytotoxicity Testing and Doubling time experiments. All tested cyPGs did not exhibit cytotoxic properties in V79 cells after 24 h of incubation calculated as the release of LDH in the cell culture medium (Figure 1A). The positive control SDS (0.1% w/v) significantly increased LDH activity in the supernatant, 5-fold in comparison to the solvent control (DMSO 0.5% v/v). In addition, no effect on the doubling time of V79 after incubation with the respective cyPGs was observed (exemplarily shown for 15dPGJ2 in Figure 1B), thus letting us exclude cytostatic effects. DNA-Damaging Properties of cyPGs. DNA-damaging properties of cyPGs were investigated in the comet assay (Figure 2). V79 cells were incubated with PGA2, PGB2, or 15dPGJ2 for 1 h in serum-free cell culture medium. The treatment of V79 cells with the redox cycler menadione at a concentration of 10 μM significantly enhanced the rate of DNA strand breaks compared to the respective solvent control. Postincubation treatment of the test samples with FPG (white striped bar) was used to screen oxidative DNA damage. A highly significant increase of FPG-sensitive sites was observed at 10 μM menadione in comparison to the rate of DNA strand breaks without FPG-treatment. In the absence of FPG, 15dPGJ2 (5 μM) induced a slight but significant increase in DNA strand breaks. At 7.5 μM, PGA2 was found to significantly enhance DNA strand breaks, whereas PGB2 exhibited no DNAdamaging properties. However, the DNA-damaging effects of the most potent analogue (15dPGJ2, 10 μM) were only minor (tail intensity, TI, < 4%) in comparison to the positive control menadione (TI > 8%). A significant increase in FPG-sensitive sites was observed after incubation of V79 cells with 0.1 μM 15dPGJ2, 1 μM PGA2, or 2.5 μM PGB2 (Figure 2). Statistically significant differences in DNA damage between cell incubations with and without FPG were found at concentrations ≥1 μM 15dPGJ2, ≥ 2.5 μM PGA2, or ≥7.5 μM PGB2. At the highest concentration tested (10 μM), the DNA strand breaking potency of cyPGs can be ranked as 15dPGJ2 > PGA2 > PGB2. However, after an enhanced incubation time of 24 h the three tested cyPGs did not exhibit DNA-damaging properties with and without FPG treatment (Figure 3). Generation of Cellular Reactive Oxygen Species (ROS). The V79 cell line was proved as a not suitable test system to measure intracellular ROS via DCF-assay. Because of the many wash steps, we saw a high cell loss; thus, no fluorescent increase including the positive control menadione could be measured. Therefore, we change the cell system to an established method, which has been previously reported by Pelka et al.27 For all cyPGs at a concentration range of 0.1 up to

Figure 1. (A) Lack of cytotoxicity measured after 24 h of incubation in the LDH leakage assay. The data are presented as the mean ± SD of at least three independent experiments, each performed in duplicate. The significances indicated refer to the significance level as compared to the respective control calculated by Student’s t test (*** = p ≤ 0.001). (B) Impact of 15dPGJ2 on the doubling time of V79 cells. The data are presented as the mean ± SD of at least two independent experiments, each performed in quadruplicate. (C) Incubation protocol of the respective doubling time experiment.

10 μM, the relative fluorescence remained constant around the level of 100% during the observation time of 3 h (Figure 4). Chemical GSH-Binding Capacity and Impact on Cellular GSH Status. Thiol reactivity of cyPGs in a cell free system was detected by measuring reduced GSH. Specifically, 15dPGJ2 as well as PGA2 exhibited comparable spontaneous reactivity with GSH (Figure 5A), whereas only a marginal sulfhydryl reactivity was observed with PGB2. Cyclopentenone was included as positive control. Furthermore, we investigated whether cyPGs also affect the total GSH (tGSH) status of intact V79 cells according to the protocol of Tietze.25 Under the applied incubation conditions, cell viability was maintained throughout the experiment at >90% determined by trypan blue exclusion (data not shown). Over an incubation time of 1 h, the assay was performed for all cyPGs at a concentration range of 0.1−10 μM as shown in 255



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Figure 2. Single cell gel electrophoresis (comet assay) with cyPG treated V79 cells. The cells were treated for one hour with the respective test compound. The redox cycler menadione (MEN) was included as a positive control. The data presented are the means ± SD of at least three independent experiments, each performed in duplicate. The significances indicated refer to the significance level compared to the respective control (Student’s t-test, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.005) or to the effect of FPG-treatment (Student’s t-test, # = p ≤ 0.05, ## = p ≤ 0.01, ### = p ≤ 0.005).

Figure 4. Generation of intracellular ROS in HT29 cells (DCF-assay). The redoxcycler menadione was included as a positive control in the assay. ROS generation is expressed as relative fluorescent units RFUs [%]. The data are presented as the mean ± SD of at least three independent experiments. The significances indicated refer to the significance level compared to the respective control (Student’s t-test, *** = p < 0.001).

Figure 3. Single cell gel electrophoresis (comet assay) with V79 cells treated with the respective cyPGs for 24h. The redox cycler menadione (MEN) was included as positive control. The data presented are the means ± SD of at least three independent experiments, each performed in duplicate. Significances indicated refer to the significance level compared to the respective control (Student’s t-test, *** = p ≤ 0.001) or to the effect of FPG-treatment (Student’s ttest, ## = p ≤ 0.01).

Micronuclei and Apoptosis Induction. V79 cells treated with cyPGs were analyzed by fluorescence microscopy with respect to micronuclei and apoptosis induction. Treatment with 0.6 μM MMC for 24 h or 0.5 μM NOC for 16 h significantly increased the number of micronucleated cells as compared to solvent-treated cells (Table 1). A significant increase in

Figure 5B. 15dPGJ2 at concentrations ≥1 μM and PGA2 at concentrations ≥5 μM were found to significantly reduce the content of tGSH in V79 cells, whereas PGB2 slightly decreased tGSH without reaching statistical significance. 256



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Table 1. Micronucleated after Treatment of V79 Cells with the Test Compounds (1 or 24 h) and after a CompoundFree Post-Incubation Period of 20 ha compound DMSO MMC NOC PGA2 PGB2 15dPGJ2

concn [μM]

treatment

micronuclei per 1000 nuclei

0.5% v/v 0.5% v/v 0.6 0.5 10 10 1 5 7.5 10 1 5 7.5 10

1h 24 h 24 h 16 h 1h 1h 1h 1h 1h 1h 24 h 24 h 24 h 24 h

9±3 7±4 108 ± 7*** 35 ± 9** 11 ± 2 10 ± 4 13 ± 7 19 ± 12 24 ± 10* 28 ± 3** 5±2 n.d. 8±3 10 ± 2

The data presented are the means ± SD of at least three independent experiments. Significances indicated refer to the significance level compared to the respective control (Student’s t-test, *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). n.d.: not determined. a

Figure 5. (A) Spontaneous GSH reactivity of the test compounds was measured photometrically at λ = 412 nm after the chemical reaction with Ellmann’s reagent. The data include a blank rate to correct for autoxidation reactions of GSH. (B) tGSH content was measured after the incubation of V79 cells under serum-free cell culture conditions with cyPGs for 1 h. As positive control (PC), L-buthionin-(S,R)sulfoxime (BSO, 1 mM) was applied. Results are shown as percent of control (DMSO 0.5% v/v). The data are presented as the mean ± SD of at least three independent experiments, each performed in duplicate. The significances indicated refer to the significance level as compared to the respective control calculated by Student’s t test (* = p ≤ 0.05, ** = p ≤ 0.01, and *** = p ≤ 0.001).

micronuclei frequency was observed after the treatment of V79 cells with 15dPGJ2 (≥7.5 μM) for 1 h, followed by a substancefree incubation period of 20 h (Table 1), whereas PGA2 and PGB2 were found to be ineffective. However, after a prolonged treatment (24 h), 15dPGJ2 also showed no effect (Table 1), in line with the results of the comet assay. Furthermore, we addressed the question whether 15dPGJ2 acts as a clastogenic or aneugenic compound. A significantly increased amount of kinetochore-positive micronucleated cells indicative for an aneugenic mode of action was observed after cell incubation with 10 μM 15dPGJ2 (Figure 6A). The ratio of kinetochore-positive versus kinetochore-negative micronuclei was almost identical to that after NOC treatment. In addition, NOC (0.5 μM) and the test compound 15dPGJ2 significantly increased the number of apoptotic cells (Figure 6B). In line with the results on micronuclei induction, no enhanced apoptosis was observed after 24 h of incubation. Lack of Mutagenicity. The spontaneous frequency of HPRT mutations in V79 cells was 12.0 ± 5.0 per 106 viable cells. The established mutagen MNNG served as the positive control. A noncytotoxic concentration of 10 μM MNNG was found to enhance the mutant frequency to 168 ± 44 per 106

Figure 6. (A) Increase of kinetochore positive micronuclei and (B) apoptotic cells after incubation with 15dPGJ2 for 1 h and a postincubation period of 20 h. Nocodazole (NOC, 0.5 μM, 16 h) was served as positive control and DMSO (0.5% v/v) as solvent control. The data are presented as the mean ± SD of at least three independent experiments. The significances indicated refer to the significance level as compared to the respective control calculated by Student’s t test (* = p ≤ 0.05; **= p ≤ 0.01).

cells (Figure 7). Mutant frequency was not induced by the treatment with any of the three cyPGs. 257



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DNA strand breaks, whereupon 15dPGJ2 was found to be the most potent. Oxidative DNA damage could be triggered directly by the formation of reactive oxygen/nitrogen species (ROS/RNS) or indirectly by perturbation of cellular redox sensitive signaling cascades such as the Nrf-2 pathway.33−36 Furthermore, it has to be considered that FPG is not limited to recognizing oxidative DNA lesions but also detects a spectrum of DNA adducts mediated by methylation.32 In contrast to our results on the human colon carcinoma cell line HT29, Wang and Mak have reported that 15dPGJ2 (10 μM) enhances ROS levels in A549 lung adenocarcinoma cells after 2 h of incubation with reaching a maximum after 4 h of incubation.14 The intracellular GSH level was determined as an indicator for enhanced oxidative stress within the cell system. cyPGs were found to affect the tGSH level in V79 cells. Of note, the extent of tGSH depletion was found to correlate with the amount of electrophilic carbons (15dPGJ2 > PGA2 > PGB2). 15dPGJ2 has been reported to decrease intracellular GSH concentration in A549 cells and in B lymphocytes.14,37 Because of their electrophilic nature, cyPGs may form Michael adducts with GSH both enzymatically, through the action of glutathione-S-transferases, and nonenzymatically.9,38,39 In our experiments, only the α,β-unsaturated moiety at the cyclopentenone ring contributed to the observed chemical reactivity of cyPGs. Likewise, cyclopentenone nearly possessed a comparable activity toward GSH. Our results are supported by previous studies, which have shown that GSH conjugation occurs exclusively at the C-9 atom of 15dPGJ2.40 In the human carcinoma cell line HepG2, GSH conjugation occurs in a timedependent manner together with a reduction of the electrophilic C9 of the eicosanoid.38 In contrast, an increase in GSH in the intracellular and extracellular compartments of different epithelial cell systems over a 48 h treatment with a cyPG mixture or the single compounds PGD2, PGJ2, and 15dPGJ2 has been reported.41 It could be postulated that the lack of genotoxicity after 24 h of incubation is associated with a switch of cellular redox response by increased GSH levels. However in our findings, it is unlikely that GSH-binding alone is responsible for the observed decrease of GSH protein levels, given that the used cyPG concentrations were in a micromolar range and that cellular GSH levels are up to 5 mM. Some mechanisms for GSH depletion by 15dPGJ2 have been discussed in the literature. Song et al. have proposed that the accumulated 15dPGJ2-GSH conjugate is pumped out via a member of the ABC transporter family, the MRP1/GS-X pump, and after a release from GSH, the lipophilic 15 dPGJ2 reenters the cell, whereas the hydrophilic GSH remains outside.42 Oxidative stress is a well understood inducer of the transcription of specific genes associated with cell response or cell death, while GSH has also been postulated as a modulator of gene transcription.22,23 Several GSH-related enzyme activities have been described to be affected by Nrf-2. cyPGs, particularly 15dPGJ2, led to a translocation or Nrf-2 into the nucleus and to the antioxidative response element (ARE)related gene transcription by cysteine modification of the Kelch-like ECH-associated protein 1 (Keap 1). In addition to Keap 1 modifications, several protein kinase pathways have been characterized to participate in Nrf2-dependent gene expression.17,36 A limited number of studies have demonstrated that mitogen activated protein kinase p38, extracellular regulated kinases ERK1/2, and protein kinase B (PKB/AKT) are involved in the induction of heme oxygenase or glutamate cysteine ligase (GCL).42−46 It could be speculated that initial

Figure 7. Mutant frequencies after treatment of V79 cells with DMSO (0.5% v/v, solvent control), the positive control MNNG, or test compounds for 1 h measured in the HPRT assay. Data represent the means of three independent experiments ± SD. Asterisks indicate significant differences to the corresponding solvent control. Levels of significance: Student’s t-test *** = p ≤ 0.001.

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DISCUSSION cyPGs have been demonstrated to inhibit proliferation of tumor cells in vitro,5,14,15 to affect signaling cascades involved in inflammation,7,16,17 and to show antiviral activity against polio, Sendai, and HIV viruses.28 However, several authors have proposed that cyPGs, notably 15dPGJ2, support the tumorigenic process.20,21 In this study, potential genotoxic properties of cyPGs were investigated. The three cyPGs PGA2, PGB2, and 15dPGJ2 were included because we postulated differences in cellular biofunctionality as a consequence of their chemical structures. 15dPGJ2 and PGA2 bear an electrophilic carbon (Chart 1) in the cyclopentenone ring. 15dPGJ2 bears another reactive carbon located in the side chain. The highly electrophilic carbon centers can serve as acceptors in a Michael addition reaction leading to irreversible alkylation of, e.g., cysteine residues in target molecules.29 In the comet assay, a low capacity of PGA2 (≥7.5 μM) and 15dPGJ2 (≥5 μM) directly inducing DNA damage was observed, whereas PGB2 did not exhibit any DNA-damaging properties. The shown DNA strand breaks may be associated with an inhibition of topoisomerase activity. Topoisomerase enzymes are responsible for the maintenance of DNA structure and conformation. These enzymes generate a covalent enzyme−DNA intermediate, the so-called cleavable complex, which creates transient DNA strands breaks, thus promoting the relaxation of supercoiled DNA. PGA2 and 15dPGJ2 have been found to decrease topoisomerase II activity in a cell free test system with IC50values of 98 μM and 22 μM, respectively.30 Another study showed that 15dPGJ2 acts as catalytic topoisomerase inhibitor without stabilizing the enzyme−DNA intermediate and does not exhibit intercalative properties.31 Overall, these findings indicate that an impact on topoisomerases may contribute to the observed DNA-damaging properties of cyPGs. A modified comet assay protocol was used for the detection of FPG sensitive sites. The bacterial repair enzyme FPG recognizes modified purine bases such as 8-oxo-guanine or 2,6diamino-4-hydroxy-5-formamidopyrimidine. Under the conditions applied in the comet assay, these apurinic sites are measured as additional DNA strand breaks. Enhanced occurrence of FPG-sensitive sites is often associated with oxidative DNA damage.32,33 All three cyPGs increased oxidative 258



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GSH depletion resulted in ROS accumulation, which leads to Nrf-2 related gene expression. Especially, the increased expression of GCL, the rate-limiting enzyme in GSH synthesis, provokes the propagation of de novo GSH synthesis.42,46 Overall, these findings support the hypothesis that the impact of cyPGs on GSH status and on its subsequent ROS-mediated Nrf-2 signaling pathway contributes to the strong formation of FPG-sensitive sites after a short time of incubation (1 h) and the reversal of DNA damage after an extended incubation time of 24 h. With respect to clastogenic/aneugenic effects, only 15dPGJ2 at concentrations ≥7.5 μM significantly induced the number of micronucleated cells. The majority of them were kinetochore positive indicating an aneugenic mode of action. The potency of 15dPJ2 was comparable to the positive control NOC. In the human nonsmall lung carcinoma A549 cell line, an induction of apoptosis after the treatment with 10 μM 15dPGJ2 has been demonstrated previously.14 In our experiments, the induction of apoptosis by 15dPGJ2 was related to the formation of micronuclei, which is in agreement with recent reports on the microtubule inhibitor NOC.47,48 It could be postulated that 15dPGJ2 may interfere with the microtubule assembly by binding to microtubular proteins. Alternatively, GSH depletion per se has been discussed either to induce or potentiate apoptosis.49,50 The possibility that micronucleated cells may be eliminated by apoptosis, thus preventing mutagenicity in the V79 HPRT test system, needs further investigations. Furthermore, the negative results in the HPRT assay could also involve large chromosome deletions.51 This hypothesis is supported by investigations showing that oxidative stress is at most weakly mutagenic in terms of point mutations and small deletions but mutagenic through a mechanism involving large rearrangements.52,53 With respect to the relevance of our findings for the in vivo situation, more information on the kinetics of cyPGs is required. Oh et al. have demonstrated that about 98% of exogenously added 15dPGJ2 can be inactivated in the cell culture medium.17 We carried out our experiments under serum free conditions because a strong interference of albumin with 15dPGJ2 has been suggested.17 Furthermore, in cells cyPGs have a short half-life due to GSH conjugation, which has been discussed as their major metabolization step.38,42,46 Hardy et al. have demonstrated that 15dPGJ2 and 15dPGJ2-like compounds are generated in vivo under conditions of oxidant stress.54 Previous studies have shown that 15dPGJ2 is synthesized during mammalian inflammatory responses and could be detected in the inflammatory exudates from carrageenan-induced pleurisy in rats (803 ± 167 pg/ mL).55−57 Preliminary in vivo data suggest that cyPGs are rapidly metabolized via conjugation with GSH and excreted into urine as GSH adducts.55 For 15dPGJ2, the reported plasma levels reached from 5 pg/mL up to about 100 pg/mL depending on pathophysiological conditions.58,59 In conclusion, cyPGs are endogenous endocrine mediators that have raised considerable interest due to their proposed anti-inflammatory and antiproliferative properties. However, little is known so far about potential negative functions such as genotoxicity. We found slight direct DNA-damaging properties after cell incubation with PGA2 and 15dPGJ2 and a strong increase in FPG-sensitive sites for all test compounds after a short time of incubation. DNA damage may be mediated via cellular oxidative stress due to the fact that PGA2 and 15dPG2

significantly decreased the cellular GSH level and showed high chemical GSH-binding capacity. Furthermore, we demonstrate that 15dPGJ2 exhibits aneugenic properties which come along with an induction of apoptosis. However, a prolonged incubation time, led to a lack of genotoxicity. Thus, let us speculate that a potential genotoxic and/or mutagenic potency of cyPGs does not become important but rather that the cellular stress response via the Nrf-2 signaling cascade, DNArepair mechanisms, or apoptotic events give a high priority. In summary, we found a potential in vitro genotoxicity of cyPGs associated with a strong impact on the cellular redox system. The shown mode of action of selected cyPGs may contribute to the association between chronic inflammation and cancer development.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49-(0)631-2054765. Fax: +49-(0)631-2054398. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Doris Marko and Professor Elke Richling for providing the FPG enzyme. REFERENCES

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