Genotoxic Properties of Cyclopentenone Prostaglandins and theOnset of Glutathione DepletionGergely 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 Universitat Kaiserslautern,Erwin-Schrodinger-Str. 52, 67663 Kaiserslautern, Germany
ABSTRACT: Prostaglandins are endogenous mediators formed from arachidonic acid by cyclooxygenases and prostaglandinsynthases 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 andcancer. However, little is known so far about possible adverse health effects of cyPGs. We addressed the question whetherselected cyPGs at a concentration range of 0.1−10 μM exhibit mutagenic and genotoxic properties in the hamster lung fibroblastV79 cell line and whether these effects are accompanied by a depletion of intracellular glutathione (GSH). The cyPGs 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) and prostaglandin A2 (PGA2) significantly induced DNA damage in V79 cells after 1 h ofincubation. 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 resultsthat 15dPGJ2 acts as an aneugenic agent which induces the amount of kinetochore positive micronuclei associated with anincrease of apoptosis. The strong potency of cyPGs to rapidly bind GSH measured in a chemical assay and to significantly reducethe GSH level after only 1 h of incubation may contribute to the observed oxidative DNA strand breaks, whereas directly inducedoxidative stress via reactive oxygen species could be excluded. However, after an extended incubation time of 24 h nogenotoxicity 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 beendemonstrated, which may be involved in carcinogenesis mediated by chronic inflammation.
■ INTRODUCTION
Cyclopentenone prostaglandins (Chart 1) are reactive lipidicmediators that arise by nonenzymatic dehydration of certainprostaglandins (PGs). PGs belong to the family of eicosanoids,being derived from arachidonic acid liberated from membranephospholipids by the action of phospholipase A2.
1,2 cyPGs arecharacterized by the presence of the electrophilic α,β-unsaturated carbonyl moiety in the cyclopentene ring
responsible for their strong potency to form Michael adducts,e.g., with GSH. The formation of covalent protein adducts isassociated with a modulation in protein function, which hasbeen described in depth in the literature.1 Transcription factorssuch as activator protein-1 (AP-1), nuclear factor κB (NfκB), or
Received: October 29, 2012Published: January 22, 2013
nuclear factor-E2-related factor 2 (Nrf-2), proteins involved inthe regulation of the cellular redox status, such as thioredoxin,thioredoxin reductase, and cytoskeletal proteins, have beencharacterized as target proteins of cyPGs.3−10 With regard tothe cell signaling responses to cyPGs, those to 15dPGJ2 are thebest understood so far. 15dPGJ2 binds as an endogenous ligandto the peroxisome proliferator-activated receptor γ(PPARγ).11,12 Furthermore, the postulated anti-inflammatoryeffects are associated with a modulation of the NF-κB pathwayvia direct inhibition of IkB kinases.13 In addition, 15dPGJ2 hasbeen demonstrated to induce apoptosis, to inhibit cell growthand differentiation and to induce the expression of antioxidantresponse element (ARE)-related genes by activating the Nrf-2pathway.14−17 However, cyPGs are generated during inflam-matory responses. Chronic inflammation, frequently combinedwith chronic infection, has been recognized in epidemiologicaland mechanistic studies to be a critical component for tumorformation and progression, being associated with carcino-genesis in one of every five cancer patients worldwide.18,19
Among the factors formed during inflammation, low-molecularweight mediators, such as cyPGs may contribute to theseeffects, particularly if they are genotoxic. 15dPGJ2 was found toinduce cell proliferation of the colorectal cancer cell line HCA-7.20 Millan et al. published that 15dPGJ2 during 7,12-dimethylbenz[a]anthracene treatment significantly increasesthe rate of formation, size, and vascularization of papilloma.21
The aim of the present study was to clarify whether selectedcyPGs (PGA2, PGB2, and 15dPGJ2) affect cellular targets suchas the antioxidative protein GSH and/or DNA. GSH is themajor endogenous thiol antioxidant, having an extensive role inthe regulation of cellular redox balance as well as thedetoxification of exogenous and endogenous substances suchas xenobiotics, ionizing radiation, or several cytokines.22,23
Therefore, the total intracellular GSH level can be used for theassessment of the cellular redox status. Genotoxic properties ofthe respective cyPGs were investigated by single cell gelelectrophoresis (comet assay), the HPRT assay, and themicronucleus assay in V79 cells. In addition, to detect oxidativeDNA damage, a modified comet assay protocol with the DNArepair enzyme formamido-pyrimidine-DNA-glycosylase (FPG)was used. Furthermore, investigations about the mode of actionof cyPG genotoxicity being aneugenic or clastogenic wereincluded.
bromide, menadione, mitomycin C (MMC), nocodazole (NOC), and6-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-phenyl-indole-dihydrochloride (DAPI) was purchased from Carl Roth
(Karlsruhe, Germany). Kaiser’s glycerol gelatin and methylene bluewere obtained from Merck (Darmstadt, Germany). Cyclopentenoneprostaglandines were purchased from Santa Cruz Biotechnology(Heidelberg, Germany).
Cell Culture. Male Chinese hamster V79 lung fibroblasts(Deutsche Sammlung fur Mikroorganismen and Zellkultur, DSMZ,Braunschweig, Germany) were grown in Dulbecco’s modified Eagle’smedium (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 with10% 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 atmospherecontaining 5% CO2 and were routinely tested for the absence ofmycoplasm contamination. Compounds were dissolved in DMSO andadded to the medium to yield a final DMSO concentration of 0.5% (v/v).
Lactate Dehydrogenase (LDH)-Leakage Assay. Cytotoxicitywas determined by measuring the lactate dehydrogenase (LDH)activity in the cell medium using a commercial LDH cytotoxicity assaykit (Cayman Chemical, Ann Arbor, MI, USA). V79 cells (2 ×104) in120 μL of serum containing medium were seeded on 96-well platesand allowed to grow for 24 h. Thereafter, cells were incubated with thetest compounds (0.1, 1, 2.5, 5. 7.5, and 10 μM) under serum-free cellculture 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 and400g, and 100 μL of the supernatant was transferred to a second 96well plate. To every plate, a standard LDH calibration line with thefollowing LDH activities (0, 62.5, 125, 250, 500, and 1,000 μU) wasincluded as duplicates. One hundred microliter LDH-reactionsolutions were added, the plate was gently shaken for 30 min at 25°C and measured immediately with a plate reader at λex = 490 nm. Theeffects on the membrane integrity were calculated as μU per mL.
Determination of Doubling Time. V79 cells (4000/well) wereseeded in 24 well plates. After 24 h, the culture medium was removed,and cells were washed with phosphate buffered saline and wereincubated with the respective cyPGs under serum free conditions for 1h. After that, the incubation medium was removed, and the cells werecultivated in serum-containing medium. This step was repeated after72 h. Cell number was counted in quadruplicate over one week everyday.
Comet Assay. The comet assay was performed according to themethod of Tice et al.24 V79 cells (1 × 106) were seeded into Petridishes (⌀ = 6 cm, 5 mL medium containing 10% FCS) and allowed togrow for 24 h prior to treatment. Cells were treated for 1 h with thesolvent control or the test compounds under serum-free conditions. Asa 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 meltingagarose and distributed onto an encoded frosted glass microscopeslide, precoated with a layer of normal melting agarose. The slideswere coverslipped and kept at 4 °C for 10 min to allow solidification ofthe agarose. After removing the cover glass, slides were immersed for 1
Chart 1. Chemical Structures of the Used cyPGs: (A) PGA2, (B) PGB2, and (C) 15dPGJ2a
h at 4 °C in lysis solution (89 mL of lysis stock solution; 2.5 mMsodium chloride, 100 mM EDTA, 10 mM Tris, and 1% w/v N-laurylsarcosin sodium salt; 1 mL of Triton-X-100 and 10 mL ofDMSO). For the additional detection of oxidative DNA damage, slideswere 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 bovineserum albumin), covered with 50 μL of either enzyme buffer or FPGenzyme, and incubated for 30 min at 37 °C. Subsequently, DNA wasallowed to unwind (300 mM NaOH and 1 mM EDTA, pH 13.5, 20min, 4 °C) followed by horizontal gel electrophoresis at 4 °C for 20min (300 mA = const). Thereafter, the slides were washed three timeswith 0.4 M Tris-HCl, pH 7.5, and stained with ethidium bromide (40μL per coverslip, 20 μg/mL). Fluorescence microscopy was performedwith a Zeiss Axioskop 50/AC (λex = 546 ± 12 nm; λem > 590 nm).Slides were subjected to computer-aided image analysis (Comet AssayIV System, Perceptive Instruments, Suffolk, Great Britain), scoring 50images per slide randomly picked. For each concentration of testcompound, 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 aspercentage of overall DNA fluorescence intensity in the respectivecell). In parallel to the comet assay, viability of the cells wasdetermined 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 of40,000 in 100 μL of serum-containing medium per well, which werepreviously allowed to grow for 48 h. Afterward, the cell culturemedium was removed, and cells were washed with phosphate bufferedsaline and incubated with 100 μL of 50 μM 2′,7′- dichlorofluorescin-diacetate solution (DCFH-DA) for 30 min at 37 °C. The dyesupernatant was removed, and the cells were washed twice withphosphate buffered saline. HT29 cells were incubated with cyPGs(0.1−10 μM). Menadione was included in the testing as a positivecontrol for the induction of ROS. In the presence of intracellular ROS,cellular esterases generated 2′,7′-dichlorofluorescin (DCFH) that wasoxidized to fluorescent 2′,7′-dichlorofluorescein (DCF). The increasein fluorescence intensity was measured with a fluorometer at λex = 485nm 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 anincubator (37 °C, 5% CO2). Cell culture medium was removed; thecells were rinsed off with 5 mL of phosphate buffered saline (PBS) andtreated for 1 h with the test substances or for 24 h with the positivecontrol mitomycin C (MMC, 0.6 μM) or with UVB (150 J/m2) toinduce apoptosis. After removing the test compounds by mediumreplacement, the cells were incubated with FCS-containing mediumfor another 20 h and washed with PBS. Cells were fixed with 3 mL ofCarnoy’s fixation solution (methanol/acetic acid 3:1) per Petri dish at−20 °C for 1 h. For staining, fixation solution was removed, and cellswere 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), washedwith ice-cold methanol, and air-dried. Petri dishes were coverslipped,and the dried cell layers were then covered with about 50 μL of meltedKaiser’s glycerol gelatin, and coverslips were fixed on top. Fluorescencemicroscopy was performed with a Zeiss Axioskop. Micronuclei andapoptotic cells were always derived from at least three independentsets of experiments and from the evaluation of 2000 individual cellsper concentration (1000/Petri dish).CREST-Staining. V79 cells were seeded onto cover slides in
quadriPERM (Sarstedt, Numbrecht, Germany) dishes. After 24 h inculture, the medium was removed, and test compounds were added for60 min, except the positive control nocodazole (NOC, 0.5 μM), whichwas incubated for 16 h. Subsequently, serum-containing medium wasadded. After 20 h, cells were fixed with ice-cold methanol (1 h, 4 °C),and the cell membranes were made permeable with acetone. After ablocking step with goat serum for 1 h, the cells were incubated withthe 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 usedas secondary antibody. Cells were washed with Sorensen-phosphatebuffer (pH 8) and stained with a DAPI/propidium iodide/antifadesolution. 103 nuclei per cover slide were counted using a Zeiss LaserAxiovert 200 microscope (Carl Zeiss AG, Gottingen, Germany).
Thiol Reactivity. The spontaneous GSH reactivity of the testcompounds was measured in a phosphate buffered system (A/B buffer1.5 mL of buffer A, 25 mM KH2PO4 and 6 mM Na2EDTA; and 8.5mL of buffer B, 125 mM K2HPO4 and 6 mM Na2EDTA) containingequimolar concentrations of glutathione (GSH) and the respective testcompound within a total volume of 1 mL. The level of remaining GSHin the sample aliquot was measured photometrically (λ = 412 nm) dueto the reaction with Ellman’s reagent [6 mM 5,5′-dithio-bis(2-nitrobenzoic acid) to 5-thio-2-nitrobenzoate].
Glutathione (GSH) Assay. The GSH assay was performed withslight modifications according to the method of Tietze.25 V79 cells (1× 106 or 5 × 105) were seeded onto Petri dishes (Ø 6 cm) and allowedto grow for 24 h. Subsequently, cells were incubated for 1 h with thetest compounds or 24 h with the synthetic amino acid L-(−)-buthionine sulfoximine (BSO), an irreversible inhibitor of the γ-glutamyl-cysteine synthetase, as positive control Cells were collected,and the viability was determined by trypan blue exclusion. After severalwashing steps with cold phosphate buffer, cells were centrifuged at180g for 10 min at 4 °C. The resulting cell pellet was resuspended in 1mL of A/B buffer (15 mL of buffer A, 125 mM KH2PO4 and 6 mMNa2EDTA; and 85 mL of buffer B, 125 mM K2HPO4 and 6 mMNa2EDTA) 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 × 10μL of this cell suspension was used for protein quantification, whereasthe remaining cell suspension was mixed with 350 μL of 10% (w/v)sulfosalicylic acid for cell lysis. Subsequently, the suspension wascentrifuged for 10 min at 4 °C to remove the protein precipitant. Forquantitative determination of total GSH (tGSH) status, 10 μL of thesupernatant were mixed with 190 μL of the tGSH mixture [164 μL ofA/B buffer, 20 μL of 6 mM 5,5′-dithio-bis(2- nitrobenzoic acid), 4 μLof 20 mM NADPH, and 2 μL of GSH reductase (GR) solution (50 U/mL)]. In this reaction, GSSG was reduced to GSH by glutathionereductase (GR) and nicotinamide adenine dinucleotide phosphate(NADPH). Finally, the tGSH level of the cell (reduced GSSG andinitial GSH) was measured photometrically (λ = 412 nm) due to thereaction with Ellman’s reagent. The tGSH content was expressed asnmol tGSH/mg protein. Therefore, the cellular protein of the cellsuspension in the GSH assay was quantified with the BCA assayaccording to the protocol of Sigma-Aldrich (Bicinchoninic AcidProtein Assay Kit). The principle of the BCA assay relies on theformation of a Cu2+‑protein complex under alkaline conditions,followed by reduction of the Cu2+ to Cu+. The amount of reduction isproportional to the protein present. Absorbance was measuredphotometrically (λ = 592 nm).
V79 Hypoxanthine Phosphoribosyltransferase (HPRT)Assay. The HPRT assay was performed according to the methodpreviously reported by our group.26 V79 cells (1 ×106) were seededonto 75 cm2
flasks in 10 mL of FCS-containing medium (10%) andkept for 24 h under standard conditions in an incubator. Then, cellculture medium was removed, and the flasks were rinsed with 5 mL ofPBS. The test substances dissolved in DMSO were diluted with FCS-free medium, and 10 mL of this solution was added to each flask. Thepositive control was treated with 20 μM MNNG. After 1 h,supernatants were removed, and 10 mL of FCS-containing mediumwas 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 trypsinsolution was added to each flask. V79 cells were suspended in 10 mLof FCS-containing medium and counted, and 1 × 106 V79 cells weretransferred 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 andincubated for another 48 h. Cells were again trypsin-treated, and afterthat, 1 × 106 cells were transferred into 15 mL of thioguanine-containing medium (500 mL of DMEM low glucose, 25 mL of FCS, 5mL of Pen/Strep, 5 mL of 100 mM sodium pyruvate solution, 0.5 mL
of 54 mM 6-thioguanine solution), each to three 75 cm2flasks. As a
vitality control (plating efficiency; PE), 240 cells each were suspendedin 10 mL of FCS-containing medium and transferred to two Petridishes (⌀ = 10 cm). After nine days, Petri dishes and incubation flaskswere rinsed with 0.9% saline, and 5 mL of ice-cold ethanol was addedto each vessel and kept for at least 15 min at −20 °C. After removal ofethanol and addition of 3 mL of methylene blue solution (0.5% inethanol), the vessels were kept for at least 30 min at −20 °C. Then,the methylene blue solution was removed. V79 cells were carefullywashed with tap water and air-dried. The colonies per vessel werecounted, and the mean (number) was calculated for each treatment.The mutation frequency MF was calculated as MF = meanflask × 240/meanplate.
■ RESULTS
Cytotoxicity Testing and Doubling time experiments.All tested cyPGs did not exhibit cytotoxic properties in V79cells after 24 h of incubation calculated as the release of LDH inthe 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 thesolvent control (DMSO 0.5% v/v). In addition, no effect on thedoubling time of V79 after incubation with the respectivecyPGs was observed (exemplarily shown for 15dPGJ2 in Figure1B), 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, or15dPGJ2 for 1 h in serum-free cell culture medium. Thetreatment of V79 cells with the redox cycler menadione at aconcentration of 10 μM significantly enhanced the rate of DNAstrand breaks compared to the respective solvent control.Postincubation treatment of the test samples with FPG (whitestriped bar) was used to screen oxidative DNA damage. Ahighly significant increase of FPG-sensitive sites was observedat 10 μM menadione in comparison to the rate of DNA strandbreaks without FPG-treatment. In the absence of FPG,15dPGJ2 (5 μM) induced a slight but significant increase inDNA strand breaks. At 7.5 μM, PGA2 was found to significantlyenhance DNA strand breaks, whereas PGB2 exhibited no DNA-damaging properties. However, the DNA-damaging effects ofthe most potent analogue (15dPGJ2, 10 μM) were only minor(tail intensity, TI, < 4%) in comparison to the positive controlmenadione (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 differencesin DNA damage between cell incubations with and withoutFPG were found at concentrations ≥1 μM 15dPGJ2, ≥ 2.5 μMPGA2, or ≥7.5 μM PGB2. At the highest concentration tested(10 μM), the DNA strand breaking potency of cyPGs can beranked as 15dPGJ2 > PGA2 > PGB2.However, after an enhanced incubation time of 24 h the
three tested cyPGs did not exhibit DNA-damaging propertieswith and without FPG treatment (Figure 3).Generation of Cellular Reactive Oxygen Species
(ROS). The V79 cell line was proved as a not suitable testsystem to measure intracellular ROS via DCF-assay. Because ofthe many wash steps, we saw a high cell loss; thus, nofluorescent increase including the positive control menadionecould be measured. Therefore, we change the cell system to anestablished method, which has been previously reported byPelka et al.27 For all cyPGs at a concentration range of 0.1 up to
10 μM, the relative fluorescence remained constant around thelevel of 100% during the observation time of 3 h (Figure 4).
Chemical GSH-Binding Capacity and Impact onCellular GSH Status. Thiol reactivity of cyPGs in a cell freesystem was detected by measuring reduced GSH. Specifically,15dPGJ2 as well as PGA2 exhibited comparable spontaneousreactivity with GSH (Figure 5A), whereas only a marginalsulfhydryl reactivity was observed with PGB2. Cyclopentenonewas included as positive control.Furthermore, we investigated whether cyPGs also affect the
total GSH (tGSH) status of intact V79 cells according to theprotocol 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 allcyPGs at a concentration range of 0.1−10 μM as shown in
Figure 1. (A) Lack of cytotoxicity measured after 24 h of incubation inthe LDH leakage assay. The data are presented as the mean ± SD of atleast three independent experiments, each performed in duplicate. Thesignificances indicated refer to the significance level as compared to therespective control calculated by Student’s t test (*** = p ≤ 0.001). (B)Impact of 15dPGJ2 on the doubling time of V79 cells. The data arepresented as the mean ± SD of at least two independent experiments,each performed in quadruplicate. (C) Incubation protocol of therespective doubling time experiment.
Figure 5B. 15dPGJ2 at concentrations ≥1 μM and PGA2 atconcentrations ≥5 μM were found to significantly reduce thecontent of tGSH in V79 cells, whereas PGB2 slightly decreasedtGSH without reaching statistical significance.
Micronuclei and Apoptosis Induction. V79 cells treatedwith cyPGs were analyzed by fluorescence microscopy withrespect to micronuclei and apoptosis induction. Treatment with0.6 μM MMC for 24 h or 0.5 μM NOC for 16 h significantlyincreased the number of micronucleated cells as compared tosolvent-treated cells (Table 1). A significant increase in
Figure 2. Single cell gel electrophoresis (comet assay) with cyPG treated V79 cells. The cells were treated for one hour with the respective testcompound. The redox cycler menadione (MEN) was included as a positive control. The data presented are the means ± SD of at least threeindependent 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 3. Single cell gel electrophoresis (comet assay) with V79 cellstreated with the respective cyPGs for 24h. The redox cyclermenadione (MEN) was included as positive control. The datapresented are the means ± SD of at least three independentexperiments, each performed in duplicate. Significances indicated referto the significance level compared to the respective control (Student’st-test, *** = p ≤ 0.001) or to the effect of FPG-treatment (Student’s t-test, ## = p ≤ 0.01).
Figure 4. Generation of intracellular ROS in HT29 cells (DCF-assay).The redoxcycler menadione was included as a positive control in theassay. ROS generation is expressed as relative fluorescent units RFUs[%]. The data are presented as the mean ± SD of at least threeindependent experiments. The significances indicated refer to thesignificance level compared to the respective control (Student’s t-test,*** = p < 0.001).
micronuclei frequency was observed after the treatment of V79cells with 15dPGJ2 (≥7.5 μM) for 1 h, followed by a substance-free incubation period of 20 h (Table 1), whereas PGA2 andPGB2 were found to be ineffective. However, after a prolongedtreatment (24 h), 15dPGJ2 also showed no effect (Table 1), inline with the results of the comet assay.Furthermore, we addressed the question whether 15dPGJ2
acts as a clastogenic or aneugenic compound. A significantlyincreased amount of kinetochore-positive micronucleated cellsindicative for an aneugenic mode of action was observed aftercell incubation with 10 μM 15dPGJ2 (Figure 6A). The ratio ofkinetochore-positive versus kinetochore-negative micronucleiwas almost identical to that after NOC treatment. In addition,NOC (0.5 μM) and the test compound 15dPGJ2 significantlyincreased the number of apoptotic cells (Figure 6B). In linewith the results on micronuclei induction, no enhancedapoptosis 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 viablecells. The established mutagen MNNG served as the positivecontrol. A noncytotoxic concentration of 10 μM MNNG wasfound to enhance the mutant frequency to 168 ± 44 per 106
cells (Figure 7). Mutant frequency was not induced by thetreatment with any of the three cyPGs.
Figure 5. (A) Spontaneous GSH reactivity of the test compounds wasmeasured photometrically at λ = 412 nm after the chemical reactionwith Ellmann’s reagent. The data include a blank rate to correct forautoxidation reactions of GSH. (B) tGSH content was measured afterthe incubation of V79 cells under serum-free cell culture conditionswith cyPGs for 1 h. As positive control (PC), L-buthionin-(S,R)-sulfoxime (BSO, 1 mM) was applied. Results are shown as percent ofcontrol (DMSO 0.5% v/v). The data are presented as the mean ± SDof at least three independent experiments, each performed in duplicate.The significances indicated refer to the significance level as comparedto the respective control calculated by Student’s t test (* = p ≤ 0.05,** = p ≤ 0.01, and *** = p ≤ 0.001).
Table 1. Micronucleated after Treatment of V79 Cells withthe Test Compounds (1 or 24 h) and after a Compound-Free Post-Incubation Period of 20 ha
compound concn [μM] treatment micronuclei per 1000 nuclei
DMSO 0.5% v/v 1 h 9 ± 30.5% v/v 24 h 7 ± 4
MMC 0.6 24 h 108 ± 7***NOC 0.5 16 h 35 ± 9**PGA2 10 1 h 11 ± 2PGB2 10 1 h 10 ± 415dPGJ2 1 1 h 13 ± 7
5 1 h 19 ± 127.5 1 h 24 ± 10*10 1 h 28 ± 3**1 24 h 5 ± 25 24 h n.d.7.5 24 h 8 ± 310 24 h 10 ± 2
aThe data presented are the means ± SD of at least three independentexperiments. Significances indicated refer to the significance levelcompared to the respective control (Student’s t-test, *, p ≤ 0.05; **, p≤ 0.01; ***, p ≤ 0.001). n.d.: not determined.
Figure 6. (A) Increase of kinetochore positive micronuclei and (B)apoptotic cells after incubation with 15dPGJ2 for 1 h and apostincubation period of 20 h. Nocodazole (NOC, 0.5 μM, 16 h)was served as positive control and DMSO (0.5% v/v) as solventcontrol. The data are presented as the mean ± SD of at least threeindependent experiments. The significances indicated refer to thesignificance level as compared to the respective control calculated byStudent’s t test (* = p ≤ 0.05; **= p ≤ 0.01).
■ DISCUSSIONcyPGs have been demonstrated to inhibit proliferation of tumorcells in vitro,5,14,15 to affect signaling cascades involved ininflammation,7,16,17 and to show antiviral activity against polio,Sendai, and HIV viruses.28 However, several authors haveproposed that cyPGs, notably 15dPGJ2, support the tumori-genic process.20,21 In this study, potential genotoxic propertiesof cyPGs were investigated. The three cyPGs PGA2, PGB2, and15dPGJ2 were included because we postulated differences incellular biofunctionality as a consequence of their chemicalstructures.15dPGJ2 and PGA2 bear an electrophilic carbon (Chart 1) in
the cyclopentenone ring. 15dPGJ2 bears another reactivecarbon located in the side chain. The highly electrophiliccarbon centers can serve as acceptors in a Michael additionreaction leading to irreversible alkylation of, e.g., cysteineresidues in target molecules.29 In the comet assay, a lowcapacity of PGA2 (≥7.5 μM) and 15dPGJ2 (≥5 μM) directlyinducing DNA damage was observed, whereas PGB2 did notexhibit any DNA-damaging properties. The shown DNA strandbreaks may be associated with an inhibition of topoisomeraseactivity. Topoisomerase enzymes are responsible for themaintenance of DNA structure and conformation. Theseenzymes generate a covalent enzyme−DNA intermediate, theso-called cleavable complex, which creates transient DNAstrands breaks, thus promoting the relaxation of supercoiledDNA. PGA2 and 15dPGJ2 have been found to decreasetopoisomerase II activity in a cell free test system with IC50-values of 98 μM and 22 μM, respectively.30 Another studyshowed that 15dPGJ2 acts as catalytic topoisomerase inhibitorwithout stabilizing the enzyme−DNA intermediate and doesnot exhibit intercalative properties.31 Overall, these findingsindicate that an impact on topoisomerases may contribute tothe observed DNA-damaging properties of cyPGs.A modified comet assay protocol was used for the detection
of FPG sensitive sites. The bacterial repair enzyme FPGrecognizes modified purine bases such as 8-oxo-guanine or 2,6-diamino-4-hydroxy-5-formamidopyrimidine. Under the condi-tions applied in the comet assay, these apurinic sites aremeasured as additional DNA strand breaks. Enhancedoccurrence of FPG-sensitive sites is often associated withoxidative DNA damage.32,33 All three cyPGs increased oxidative
DNA strand breaks, whereupon 15dPGJ2 was found to be themost potent. Oxidative DNA damage could be triggereddirectly by the formation of reactive oxygen/nitrogen species(ROS/RNS) or indirectly by perturbation of cellular redoxsensitive signaling cascades such as the Nrf-2 pathway.33−36
Furthermore, it has to be considered that FPG is not limited torecognizing oxidative DNA lesions but also detects a spectrumof DNA adducts mediated by methylation.32 In contrast to ourresults on the human colon carcinoma cell line HT29, Wangand Mak have reported that 15dPGJ2 (10 μM) enhances ROSlevels in A549 lung adenocarcinoma cells after 2 h of incubationwith reaching a maximum after 4 h of incubation.14
The intracellular GSH level was determined as an indicatorfor enhanced oxidative stress within the cell system. cyPGswere found to affect the tGSH level in V79 cells. Of note, theextent of tGSH depletion was found to correlate with theamount of electrophilic carbons (15dPGJ2 > PGA2 > PGB2).15dPGJ2 has been reported to decrease intracellular GSHconcentration in A549 cells and in B lymphocytes.14,37 Becauseof their electrophilic nature, cyPGs may form Michael adductswith GSH both enzymatically, through the action ofglutathione-S-transferases, and nonenzymatically.9,38,39 In ourexperiments, only the α,β-unsaturated moiety at the cyclo-pentenone ring contributed to the observed chemical reactivityof cyPGs. Likewise, cyclopentenone nearly possessed acomparable activity toward GSH. Our results are supportedby previous studies, which have shown that GSH conjugationoccurs exclusively at the C-9 atom of 15dPGJ2.
40 In the humancarcinoma cell line HepG2, GSH conjugation occurs in a time-dependent manner together with a reduction of the electro-philic C9 of the eicosanoid.38 In contrast, an increase in GSH inthe intracellular and extracellular compartments of differentepithelial cell systems over a 48 h treatment with a cyPGmixture or the single compounds PGD2, PGJ2, and 15dPGJ2has been reported.41 It could be postulated that the lack ofgenotoxicity after 24 h of incubation is associated with a switchof 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 proteinlevels, given that the used cyPG concentrations were in amicromolar range and that cellular GSH levels are up to 5 mM.Some mechanisms for GSH depletion by 15dPGJ2 have beendiscussed in the literature. Song et al. have proposed that theaccumulated 15dPGJ2-GSH conjugate is pumped out via amember of the ABC transporter family, the MRP1/GS-Xpump, and after a release from GSH, the lipophilic 15 dPGJ2 re-enters the cell, whereas the hydrophilic GSH remains outside.42
Oxidative stress is a well understood inducer of thetranscription of specific genes associated with cell response orcell death, while GSH has also been postulated as a modulatorof gene transcription.22,23 Several GSH-related enzymeactivities have been described to be affected by Nrf-2. cyPGs,particularly 15dPGJ2, led to a translocation or Nrf-2 into thenucleus and to the antioxidative response element (ARE)-related gene transcription by cysteine modification of theKelch-like ECH-associated protein 1 (Keap 1). In addition toKeap 1 modifications, several protein kinase pathways havebeen characterized to participate in Nrf2-dependent geneexpression.17,36 A limited number of studies have demonstratedthat mitogen activated protein kinase p38, extracellularregulated kinases ERK1/2, and protein kinase B (PKB/AKT)are involved in the induction of heme oxygenase or glutamatecysteine 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 testcompounds for 1 h measured in the HPRT assay. Data represent themeans of three independent experiments ± SD. Asterisks indicatesignificant differences to the corresponding solvent control. Levels ofsignificance: Student’s t-test *** = p ≤ 0.001.
GSH depletion resulted in ROS accumulation, which leads toNrf-2 related gene expression. Especially, the increasedexpression 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 impactof cyPGs on GSH status and on its subsequent ROS-mediatedNrf-2 signaling pathway contributes to the strong formation ofFPG-sensitive sites after a short time of incubation (1 h) andthe reversal of DNA damage after an extended incubation timeof 24 h.With respect to clastogenic/aneugenic effects, only 15dPGJ2
at concentrations ≥7.5 μM significantly induced the number ofmicronucleated cells. The majority of them were kinetochorepositive indicating an aneugenic mode of action. The potencyof 15dPJ2 was comparable to the positive control NOC. In thehuman nonsmall lung carcinoma A549 cell line, an induction ofapoptosis after the treatment with 10 μM 15dPGJ2 has beendemonstrated previously.14 In our experiments, the inductionof apoptosis by 15dPGJ2 was related to the formation ofmicronuclei, which is in agreement with recent reports on themicrotubule inhibitor NOC.47,48 It could be postulated that15dPGJ2 may interfere with the microtubule assembly bybinding to microtubular proteins. Alternatively, GSH depletionper se has been discussed either to induce or potentiateapoptosis.49,50
The possibility that micronucleated cells may be eliminatedby apoptosis, thus preventing mutagenicity in the V79 HPRTtest system, needs further investigations. Furthermore, thenegative results in the HPRT assay could also involve largechromosome deletions.51 This hypothesis is supported byinvestigations showing that oxidative stress is at most weaklymutagenic in terms of point mutations and small deletions butmutagenic through a mechanism involving large rearrange-ments.52,53
With respect to the relevance of our findings for the in vivosituation, more information on the kinetics of cyPGs isrequired. Oh et al. have demonstrated that about 98% ofexogenously added 15dPGJ2 can be inactivated in the cellculture medium.17 We carried out our experiments underserum free conditions because a strong interference of albuminwith 15dPGJ2 has been suggested.17 Furthermore, in cellscyPGs have a short half-life due to GSH conjugation, which hasbeen discussed as their major metabolization step.38,42,46 Hardyet al. have demonstrated that 15dPGJ2 and 15dPGJ2-likecompounds are generated in vivo under conditions of oxidantstress.54 Previous studies have shown that 15dPGJ2 issynthesized during mammalian inflammatory responses andcould be detected in the inflammatory exudates fromcarrageenan-induced pleurisy in rats (803 ± 167 pg/mL).55−57 Preliminary in vivo data suggest that cyPGs arerapidly metabolized via conjugation with GSH and excretedinto urine as GSH adducts.55 For 15dPGJ2, the reported plasmalevels reached from 5 pg/mL up to about 100 pg/mLdepending on pathophysiological conditions.58,59
In conclusion, cyPGs are endogenous endocrine mediatorsthat have raised considerable interest due to their proposedanti-inflammatory and antiproliferative properties. However,little is known so far about potential negative functions such asgenotoxicity. We found slight direct DNA-damaging propertiesafter cell incubation with PGA2 and 15dPGJ2 and a strongincrease in FPG-sensitive sites for all test compounds after ashort time of incubation. DNA damage may be mediated viacellular oxidative stress due to the fact that PGA2 and 15dPG2
significantly decreased the cellular GSH level and showed highchemical GSH-binding capacity. Furthermore, we demonstratethat 15dPGJ2 exhibits aneugenic properties which come alongwith an induction of apoptosis. However, a prolongedincubation time, led to a lack of genotoxicity. Thus, let usspeculate that a potential genotoxic and/or mutagenic potencyof cyPGs does not become important but rather that thecellular stress response via the Nrf-2 signaling cascade, DNA-repair mechanisms, or apoptotic events give a high priority. Insummary, we found a potential in vitro genotoxicity of cyPGsassociated with a strong impact on the cellular redox system.The shown mode of action of selected cyPGs may contribute tothe association between chronic inflammation and cancerdevelopment.
■ ACKNOWLEDGMENTSWe thank Professor Doris Marko and Professor Elke Richlingfor providing the FPG enzyme.
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