A short chain synthetic analogue of lipid hydroperoxides was used to overload
glutathione peroxidase (GPx) in human choriocarcinoma cell line JAR cells. Cells
exposed to 100 &#181;M tBuOOH displayed a 40% reduction in ATP level and significantly
increased in membrane permeability, visualised by the lactate dehydrogenase
(LDH) release into the extracellular medium. The intracellular level of
oxygen free radicals measured as an oxidation of the dichlorodihydro-fluorescein
diacetate (H2DCF-DA) significantly increased after 2 hours of cell exposition to
100 &#181;M tBuOOH. Concomitantly MDA, 4-HNE level increased to 2 nmol/mg of
cell protein after 2 hours. Mitochondria stained with MitoTracker Red CMXRos
displayed a filamentous appearance in control cells but changed into granular
less energised organelles after exposition to tBuOOH. Collectively, the above
results indicate the importance of the contribution of oxidative stress in the
development of pre-eclampsia

A, Hallmann

J, Klimek

JH, Spodnik

M, Szkatuła

M, Woźniak

R, Milczarek

English

Via Medica Journals

Folia Morphol. Vol. 64, No. 4, pp. 304–308Copyright © 2005 Via MedicaISSN 0015–5659www.fm.viamedica.plO R I G I N A L  A R T I C L E304Address for correspondence: Prof. Jerzy Klimek, MD, PhD, Department of Pharmaceutical Biochemistry, Medical University of Gdańsk,ul. Dębinki 1, 80–211 Gdańsk, Poland, tel/fax: +48 58 349 14 60, e-mail: klimekj@amg.gda.plMimicking of glutathione peroxidasedeficiency by exposition of JAR cellsto increased level of synthetic hydroperoxideAnna Hallmann1, Ryszard Milczarek1, Michał Szkatuła3, Michał Woźniak3,Jan Henryk Spodnik2, Jerzy Klimek11Department of Pharmaceutical Biochemistry, Medical University of Gdańsk, Poland2Department of Anatomy and Neurobiology, Medical University of Gdańsk, Poland3Department of Medical Chemistry, Medical University of Gdańsk, Poland[Received 15 July 2005; Revised 27 September 2005; Accepted 27 September 2005]A short chain synthetic analogue of lipid hydroperoxides was used to overloadglutathione peroxidase (GPx) in human choriocarcinoma cell line JAR cells. Cellsexposed to 100 µM tBuOOH displayed a 40% reduction in ATP level and signi-ficantly increased in membrane permeability, visualised by the lactate dehydro-genase (LDH) release into the extracellular medium. The intracellular level ofoxygen free radicals measured as an oxidation of the dichlorodihydro-fluoresce-in diacetate (H2DCF-DA) significantly increased after 2 hours of cell exposition to100 µM tBuOOH. Concomitantly MDA, 4-HNE level increased to 2 nmol/mg ofcell protein after 2 hours. Mitochondria stained with MitoTracker Red CMXRosdisplayed a filamentous appearance in control cells but changed into granularless energised organelles after exposition to tBuOOH. Collectively, the aboveresults indicate the importance of the contribution of oxidative stress in thedevelopment of pre-eclampsia.Key words: mitochondria, choriocarcinoma, oxidative stress,pre-eclampsiaINTRODUCTIONLipid hydroperoxides (LOOH) are ubiquitous com-ponents of biological membrane. Placental tropho-blast cells are recognised as the main source of en-dogenous LOOH [16]. A deficiency of placental glu-tathione peroxidase (GPx) activity can possibly in-crease the level of tissue peroxides being stimulato-ry to the formation of placental thromboxane [6, 23]and LOOH [25] and ultimately contributing to thedevelopment of pre-eclampsia. Hydroperoxides ap-pear to play a significant role in lipid peroxidation,being both a product of the initial phase of lipidperoxidation as well as a main substrate for its fur-ther propagation. The aim of the present study wasto inspect the effect of a short chain synthetic per-oxide, namely tert-butylhydroperoxide (tBuOOH), onthe energetic state of choriocarcinoma cells, the gen-eration of free radicals inside the cell and the mor-phology of mitochondria as investigated by confocalmicroscopy.MATERIAL AND METHODSCell lineHuman choriocarcinoma cells JAR (ATCC HTB-144)were routinely cultured in 75-cm2 plastic flasks(Sarsted) in RMPI 1640 containing 1 mM sodiumpyruvate, 10 mM HEPES, 2 mM L-glutamine, 4.5 g/Lglucose, supplemented with 10% (v/v) heat-inacti-vated foetal bovine serum, 100 IU/ml penicillin and305Anna Hallmann et al., tBuOOH induced oxidative stress100 mg/ml streptomycin. The cells were grown ina humidified atmosphere with 5% CO2 at 37°C. Theywere then passaged weekly by trypsin 0.25% andEDTA 0.03%.ReagentsRMPI 1640 and tBuOOH were purchased fromSigma Aldrich Chemical (USA). The foetal bovine se-rum was produced by Gibco BRL (Grand Island, NY,USA). Trypsin with EDTA and phosphate buffer sa-line (PBS) pH 7.4 were products of Sigma Chemicals(USA). Mito Tracker Red CMXRos (CMXRos) Molecu-lar Probes, Inc. FITC — Phalloidin was produced bySigma P5282 (USA). Formaldehyde and glutaralde-hyde were purchased from Merck (Germany), Per-ma Fluor mounting solution from Immunon (Pitts-burgh, PA, USA) and Triton X-100 from Sigma Ald-rich Chemical (USA).Treatment of cells with tert-butylhydroperoxideCells were plated in 6-well plates at a concentra-tion of 5 × 105–1 × 106 cells/well (0.5–1 mg protein//ml/well). The cells were exposed to 100 µM tBuOOHat specific times (control = 0 h, 1 h, 2 h).Viability assayCell viability was evaluated by lactate dehydro-genase (LDH) release. After substracting the relevantvolume of medium from above the cells (to be usedas reference in the following steps), the cells werelysed by the addition of Triton X-100 (final concen-tration in culture dish 1%). The aliquots of the lysedcells as well as the reference culture medium werecentrifuged at 1500 × g for 3 min and supernatantswere used for the assay. The assay solution was add-ed to a cuvette containing 0.05 M Tris-HCl buffer,pH 7.4, 1 mM sodium pyruvate and 0.15 mM NADH,and spectrophotometrical analysis was performedat 340 nm. LDH release was expressed as a percent-age of total release, the release, that is, of LDH aftercell lysis by 10% Triton X-100 [9, 13].Measurement of intracellular ATP levelSample preparation: Cells (~2 × 106 cells/ml) werecollected by trypsinisation, centrifuged together withthose floating in the culture medium and washed withPBS. The cells were then suspended in cold 1.4 M HClO4and incubated for 15 min on ice to extract cellularnucleotides. Acid extracts were centrifuged (14,000RCF — 5 min), transferred to new tubes and neutra-lised to pH 6.5 by the addition of cold 3 M K3PO4prior to centrifugation (14,000 RCF — 5 min). Thesupernatants were subjected to further analysisby HPLC. The method employed for HPLC determi-nation of the intracellular level of ATP was based onthat described by Smoleński et al. [21]. Protein con-centration was evaluated using the Bradford meth-od [4] after dissolving the perchloric acid precipi-tates with 0.5 M NaOH.Staining and Visualisation of Mitochondria byConfocal MicroscopyCells growing on 24 × 24 mm-glass coverslipsunder various experimental conditions were incubat-ed with 100 nM Mito Tracker Red CMXRos (CMXRos;Molecular Probes, Inc.) for 30 minutes at 37°C in anatmosphere of 5% CO2. The cells were fixed witha fixative containing 2% glutaraldehyde and 2%formaldehyde in PBS for 30 minutes at room tem-perature and then washed in PBS and permeabilisedwith 0.1% Triton X-100 in PBS for 10 minutes at roomtemperature. This was followed by 4 washes in PBS.The coverslips with the cells were placed on glassslides in a Perma Fluor mounting solution (Immu-non, Pittburg, PA) and analysed by a confocal sys-tem Radiance 2100 (Bio-Rad, UK), equipped withKrypton/Argon lasers and mounted on an Eclipse 600(Nikon, Japan) microscope, using the software La-serSharp 2000 version 4.0 (Bio-Rad, UK) as describedpreviously [11].Flow cytometric determination of ReactiveOxygen Species and light scatterLevels of Reactive Oxygen Species (ROS) weremeasured by flow cytometry as the fluorescence ofDCF, which is the oxidation product of H2DCF-DAwith a sensitivity to ROS (H2O2, ROOH, NO). Cells wereincubated for 30 min with the probe (20 mM) at 37°C.Cell samples were then fixed (1% paraformaldehyde),cooled (4°C), and protected from light for later ana-lysis (cold-fixed cell samples). A FACSCAN (CoulterElite) flow cytometer was applied to measure theROS levels in the cells. Signals were obtained usinga 530-nm bandpass filter (FL-1 channel) for DCF. Eachdetermination was based on a mean fluorescenceintensity of 7,000 cells [1, 20].Measurement of lipid peroxidation productsCells (~10 × 106 cells/ml) were collected bytrypsinisation, centrifuged and washed with PBS.They were then suspended in PBS and the concen-tration of lipid peroxidation products measured.Malondialdehyde (MDA) and 4-hydroxynonenal(4-HNE) were measured using a lipid peroxidation306Folia Morphol., 2005, Vol. 64, No. 4assay kit (Calbiochem, San Diego, CA) [7]. Proteinconcentration was evaluated [4] and the lipid perox-idation products were described as MDA, 4-HNE/mgprotein.RESULTSEffect of tBuOOH on intracellular levels of ATPand LDH releaseTime-dependent changes of intracellular levelsof the ATP in tBuOOH-treated cells are shown inFigure 1. Two hours after the treatment, ATP lev-els had decreased to about 60% of the controlvalue (control ª 12 nmol/mg protein) (Fig. 1). Wenoticed an increase in the permeability of the cellmembrane to LDH, which was already distinct2 hours after cell treatment (Fig. 1) and increasedcontinuously in an extracellular medium thereafterto approximately 40% after 6 hours (result notshown).Effect of tBuOOH on morphological changes ofthe mitochondria in choriocarcinoma cellsThe mitochondria in the control cells stained withfluorescent dye CMXRos showed thin filamentaousstructures (Fig. 2A) under a confocal laser micro-scope. JAR cells exposed to 100 µM tBuOOH for2 hours (Fig. 2B) showed a mixed population of gra-nular mitochondria of different sizes, in many casesdistinctly enlarged. Exposition of cells to tBuOOH alsoresulted in notable changes in the shape of the cellsas well as in perturbation of the plasma membraneintegrity (Fig. 2B).Figure 1. LDH release and the concentration of ATP in JAR cellsafter treatment with 100 mM tBuOOH. The values representmeans ± SEM from 3 independent experiments.Figure 2. Morphological changes of mitochondria in JAR cells after treatment with 100 µM tBuOOH. A. Control cells; B. Treated with100 µM tBuOOH for 2 hours.307Anna Hallmann et al., tBuOOH induced oxidative stressEffect of tBuOOH on the rate of generation ofROS from choriocarcinoma cellsA typical example of a flow cytometric histogramobtained from untreated control and tBuOOH-treatedJAR cells is shown in Figure 3. A remarkable shift inthe peak intensity of DCF to the right was alreadydetected at 2 hours of tBuOOH treatment, indicat-ing that intracellular levels of ROS were distinctlyincreased by tBuOOH treatment. As a consequenceof the significant generation of ROS, an elevated levelof MDA and 4-HNE (10-fold) was observed after2 hours of cells exposition to tBuOOH (Fig. 4).DISCUSSIONWe have shown in the present study that thetBuOOH-substance with well defined pro-oxidantproperties induces mitochondrial stress in humanchoriocarcinoma cells. Generation of free radical spe-cies from tBuOOH was noticed after a 2-hour exposi-tion of cells to the pro-oxidant. This indicates an im-balance in antioxidant defence, in which glutathioneperoxidase plays a significant role, and the genera-tion of ROS. Mitochondria are the main intracellularsource of ATP and ROS [10, 15]. It has been demon-strated that mitochondria are mainly responsible forthe biotransformation of tBuOOH into free radicalproducts [12, 17]. As the result of an imbalance be-tween the generation of ROS and antioxidant defence,cellular energetic catastrophe is created and the in-tracellular ATP level decreases abruptly, reaching 60%as early as 2 hours after the treatment. As a conse-quence of the depletion of ATP, choriocarcinoma cellsstart to lose the integrity of the plasma membraneand LDH starts to escape from the cell. In our experi-mental conditions, the increased level of the hydrop-eroxides definitively overwhelms the capacity of pla-centa glutathione peroxidase, the enzyme which ismanly responsible for the “safe” two-electron reduc-tion of hydroperoxides. Under these circumstancesone-electron oxidation or a reduction in tBuOOH maycreate either the peroxyl tBuOO• or alkoxyl tBuO• rad-ical respectively [5]. The mechanism for the reactionof cytochrome c with tBuOOH was investigated byBarr and Mason by ESR spin trapping using DMPO[3]. From these analyses it was concluded that thealkoxyl radical of the hydroperoxide was the initialradical product. Lipid peroxidation is thought to beinvolved in the aetiologies of various diseases includ-ing pre-eclampsia [18, 22, 24]. So far, phospholipidhydroperoxides have been thought to be cytotoxicbecause phospholipase A2 specifically releases fattyacid hydroperoxides from phospholipids [14, 19].Transgenic mice over-expressing GPx1 have beenfound to be partially protected from the lethal effectof anti CD95, underlining the importance of GPx1 andelevated peroxide formation in apoptotic signalling[2, 8]. According to Walsh and Wang, pre-eclamptictrophoblast cells produce about 900 pmol more per-oxides than normal cells [23]. We noticed an increasedlevel of MDA and 4-HNA as early 2 hours from theincubation of the choriocarcinoma cells with 100 µMFigure 3. Changes in the intracellular levels of reactive oxygenspecies in JAR cells after treatment with 100 mM tBuOOH.Figure 4. Changes in the intracellular levels of lipid peroxidationproducts (MDA, 4-HNE) in JAR cells after treatment with 100 mMtBuOOH. The values represent means ± SEM from 3 indepen-dent experiments.308Folia Morphol., 2005, Vol. 64, No. 4tBuOOH. The above results raise the possibility of theparticipation of endogenous lipid peroxides in thedisturbance of hydroperoxide metabolism. On thebasis of the results obtained we propose that a natu-rally occurring rise in hydroperoxides may contributeto the development of pre-eclampsia.REFERENCES1. Afri M, Frimer AA, Cohen Y (2004) Active oxygen che-mistry within the liposomal bilayer. Part IV: Locating2',7'-dichlorofluorescein (DCF), 2',7'-dichlorodihydrof-luorescein (DCFH) and 2',7'-dichlorodihydrofluoresce-in diacetate (DCFH-DA) in the lipid bilayer. Chem PhysLipids, 131: 123–133.2. Bajt ML, Ho YS, Vonderfecht SL, Jaeschke H (2002) Reac-tive oxygen as modulator of TNF and fas receptor-me-diated apoptosis in vivo: studies with glutathione perox-idase-deficient mice. Antioxid Redox Signal, 4: 733–740.3. Barr DP, Mason RP (1995) Mechanism of radical pro-duction from the reaction of cytochrome c with or-ganic hydroperoxides. An ESR spin trapping investiga-tion. J Biol Chem, 270: 12709–12716.4. Bradford MM (1976) A rapid and sensitive method forthe quantitation of microgram quantities of proteinutilizing the principle of protein-dye binding. Anal Bio-chem, 72: 248–254.5. Davies MJ (1989) Detection of peroxyl and alkoxyl rad-icals produced by reaction of hydroperoxides with ratliver microsomal fractions. Biochem J, 257: 603–606.6. Eisenmann CJ, Miller RK (1995) The effect of seleniumcompounds (selenite, selenate, ebselen) on the pro-duction of thromboxane and prostacyclin by the hu-man term placenta in vitro. Toxicol Appl Pharmacol,135: 18–24.7. Esterbauer H, Cheeseman KH (1990) Determination ofaldehydic lipid peroxidation products: malonaldehydeand 4-hydroxynonenal. Methods Enzymol, 186: 407–421.8. Gouaze V, Andrieu-Abadie N, Cuvillier O, Malagarie--Cazenave S, Frisach MF, Mirault ME, Levade T (2002)Glutathione peroxidase-1 protects from CD95-inducedapoptosis. J Biol Chem, 277: 42867–42874.9. Gretzer C, Thomsen P (2000) Secretion of IL-1 andH2O2 by human mononuclear cells in vitro. Biomate-rials, 21: 1047–1055.10. Karbowski M, Kurono C, Wozniak M, Ostrowski M,Teranishi M, Nishizawa Y, Usukura J, Soji T, Wakaba-yashi T (1999) Free radical-induced megamitochon-dria formation and apoptosis. Free Radic Biol Med,26: 396–409.11. Karbowski M, Spodnik JH, Teranishi M, Wozniak M,Nishizawa Y, Usukura J, Wakabayashi T (2001) Oppo-site effects of microtubule-stabilizing and microtubule-destabilizing drugs on biogenesis of mitochondria inmammalian cells. J Cell Sci, 114: 281–291.12. Kennedy CH, Church DF, Winston GW, Pryor WA (1992)tert-Butyl hydroperoxide-induced radical production inrat liver mitochondria. Free Radic Biol Med, 12: 381–387.13. Kim JS, Southard JH (1999) Membrane stabilizingeffects of calcium and taxol during the cold storageof isolated rat hepatocytes. Transplantation, 68:938–943.14. van Kuijk FJ, Handelman GJ, Dratz EA (1985) Consecu-tive action of phospholipase A2 and glutathione per-oxidase is required for reduction of phospholipid hy-droperoxides and provides a convenient method todetermine peroxide values in membranes. J Free RadicBiol Med, 1: 421–427.15. Lee HC, Wei YH (2000) Mitochondrial role in life anddeath of the cell. J Biomed Sci, 7: 2–15.16. Maseki M, Nishigaki I, Hagihara M, Tomoda Y, Yagi K(1981) Lipid peroxide levels and lipids content of se-rum lipoprotein fractions of pregnant subjects with orwithout pre-eclampsia. Clin Chim Acta, 115: 155–161.17. O’Donnell V, Burkitt MJ (1994) Mitochondrial meta-bolism of hydroperoxide to free radicals in human en-dothelian cells: an electron spin resonance spin-trap-ping investigation. Biochem J, 304: 707–713.18. Poranen AK, Ekblad U, Uotila P, Ahotupa M (1996)Lipid peroxidation and antioxidants in normal and pre-eclamptic pregnancies. Placenta, 17: 401–405.19. Sevanian A, Wratten ML, McLeod LL, Kim E (1988)Lipid peroxidation and phospholipase A2 activity inliposomes composed of unsaturated phospholipids:a structural basis for enzyme activation. Biochim Bio-phys Acta, 961: 316–327.20. Sharikabad MN, Ostbye KM, Lyberg T, Brors O (2001)Effect of extracellular Mg2+ on ROS and Ca2+ accu-mulation during reoxygenation of rat cardiomyocytes.Am J Physiol Heart Circ Physiol, 280: 344–353.21. Smoleński RT, Lachno DR, Ledingham SJ, Yacoub MH(1990) Determination of sixteen nucleotides, nucleo-sides and bases using high-performance liquid chro-matography and its application to the study of purinemetabolism in hearts for transplantation. J Chro-matogr, 527: 414–420.22. Walsh SW (1994) The role of fatty acid peroxidationand antioxidant status in normal pregnancy and inpregnancy complicated by preeclampsia. World RevNutr Diet, 76:114–118.23. Walsh SW, Wang Y (1995) Trophoblast and placentalvillous core production of lipid peroxides, thrombo-xane, and prostacyclin in preeclampsia. J Clin Endo-crinol Metab, 80: 1888–1893.24. Walsh SW (1998) Maternal-placental interactions ofoxidative stress and antioxidants in preeclampsia. SeminReprod Endocrinol, 16: 93–104.25. Wang YP, Walsh SW, Guo JD, Zhang JY (1991) Theimbalance between thromboxane and prostacyclin inpreeclampsia is associated with an imbalance betweenlipid peroxides and vitamin E in maternal blood. Am JObstet Gynecol, 165: 1695–1700.

Mimicking of glutathione peroxidase deficiency by exposition of JAR cells to increased level of synthetic hydroperoxide

https://journals.viamedica.pl/folia_morphologica/article/download/16145/12783

Mimicking of glutathione peroxidase deficiency by exposition of JAR cells to increased level of synthetic hydroperoxide

Abstract

Similar works

Full text

Available Versions

Via Medica Journals