Reactive Oxygen Species Controls Endometriosis Progression
2009; Elsevier BV; Volume: 175; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2009.080804
ISSN1525-2191
AutoresCharlotte Ngô, Christiane Chéreau, Carole Nicco, Bernard Weill, Charles Chapron, Frédéric Batteux,
Tópico(s)Estrogen and related hormone effects
ResumoEndometriosis is associated with chronic inflammation, and reactive oxygen species (ROS) are proinflammatory mediators that modulate cell proliferation. We have investigated whether the dysregulation of ROS production in endometriotic cells correlates with a pro-proliferative phenotype and can explain the spreading of this disease. Stromal and epithelial cells were purified from ovarian endometrioma and eutopic endometrium from 14 patients with endometriosis to produce four primary cell lines from each patient. ROS production, detoxification pathways, cell proliferation, and mitogen-activated protein kinase pathway activation were studied and compared with epithelial and stromal cell lines from 14 patients without endometriosis. Modulation of the proliferation of endometriosis by N-acetyl-cysteine, danazol, and mifepristone was tested in vitro and in 28 nude mice implanted with endometriotic tissue of human origin. Endometriotic cells displayed higher endogenous oxidative stress with an increase in ROS production, alterations in ROS detoxification pathways, and a drop in catalase levels, as observed for tumor cells. This increase in endogenous ROS correlated with increased cellular proliferation and activation of ERK1/2. These phenomena were abrogated by the antioxidant molecule N-acetyl-cysteine both in vitro and in a mouse model of endometriosis. Human endometriotic cells display activated pERK, enhanced ROS production, and proliferative capability. Our murine model shows that antioxidant molecules could be used as safe and efficient treatments for endometriosis. Endometriosis is associated with chronic inflammation, and reactive oxygen species (ROS) are proinflammatory mediators that modulate cell proliferation. We have investigated whether the dysregulation of ROS production in endometriotic cells correlates with a pro-proliferative phenotype and can explain the spreading of this disease. Stromal and epithelial cells were purified from ovarian endometrioma and eutopic endometrium from 14 patients with endometriosis to produce four primary cell lines from each patient. ROS production, detoxification pathways, cell proliferation, and mitogen-activated protein kinase pathway activation were studied and compared with epithelial and stromal cell lines from 14 patients without endometriosis. Modulation of the proliferation of endometriosis by N-acetyl-cysteine, danazol, and mifepristone was tested in vitro and in 28 nude mice implanted with endometriotic tissue of human origin. Endometriotic cells displayed higher endogenous oxidative stress with an increase in ROS production, alterations in ROS detoxification pathways, and a drop in catalase levels, as observed for tumor cells. This increase in endogenous ROS correlated with increased cellular proliferation and activation of ERK1/2. These phenomena were abrogated by the antioxidant molecule N-acetyl-cysteine both in vitro and in a mouse model of endometriosis. Human endometriotic cells display activated pERK, enhanced ROS production, and proliferative capability. Our murine model shows that antioxidant molecules could be used as safe and efficient treatments for endometriosis. Endometriosis is a chronic disease characterized by the presence of endometrial tissue outside of the uterine cavity.1Giudice LC Kao LC Endometriosis.Lancet. 2004; 364: 1789-1799Abstract Full Text Full Text PDF PubMed Scopus (2561) Google Scholar It affects about 10% of women in their reproductive years and causes pelvic pain and infertility.1Giudice LC Kao LC Endometriosis.Lancet. 2004; 364: 1789-1799Abstract Full Text Full Text PDF PubMed Scopus (2561) Google Scholar The pathophysiology of endometriosis remains poorly understood. The main theory on the pathogenesis of endometriosis, proposed in the 1920s, is the retrograde menstruation of endometrial tissue through fallopian tubes into the peritoneal cavity.1Giudice LC Kao LC Endometriosis.Lancet. 2004; 364: 1789-1799Abstract Full Text Full Text PDF PubMed Scopus (2561) Google Scholar The development of the disease in the pelvis is attributed to the attachment and the survival of endometrial cells in the peritoneal cavity, progressive invasion of the peritoneum, with neoangiogenesis leading to the spreading of the disease.1Giudice LC Kao LC Endometriosis.Lancet. 2004; 364: 1789-1799Abstract Full Text Full Text PDF PubMed Scopus (2561) Google Scholar, 2Healy DL Rogers PA Hii L Wingfield M Angiogenesis: a new theory for endometriosis.Hum Reprod Update. 1998; 4: 736-740Crossref PubMed Scopus (153) Google Scholar Several mechanisms such as metaplasia of the coelomic epithelium, environmental factors, genetic predisposition, immunological abnormalities, and chronic inflammation have been proposed to explain the pelvic development of endometriosis.1Giudice LC Kao LC Endometriosis.Lancet. 2004; 364: 1789-1799Abstract Full Text Full Text PDF PubMed Scopus (2561) Google Scholar, 3Vinatier D Dufour P Oosterlynck D Immunological aspects of endometriosis.Hum Reprod Update. 1996; 2: 371-384Crossref PubMed Scopus (87) Google Scholar Indeed, proinflammatory cytokines and growth factors were found to be elevated in the peritoneal fluid of women with endometriosis and could contribute to the proliferation of endometriotic implants and neoangiogenesis.2Healy DL Rogers PA Hii L Wingfield M Angiogenesis: a new theory for endometriosis.Hum Reprod Update. 1998; 4: 736-740Crossref PubMed Scopus (153) Google Scholar, 4Sugawara J Fukaya T Murakami T Yoshida H Yajima A Increased secretion of hepatocyte growth factor by eutopic endometrial stromal cells in women with endometriosis.Fertil Steril. 1997; 68: 468-472Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 5Donnez J Smoes P Gillerot S Casanas-Roux F Nisolle M Vascular endothelial growth factor (VEGF) in endometriosis.Hum Reprod. 1998; 13: 1686-1690Crossref PubMed Scopus (459) Google Scholar Chronic inflammation has also been associated with increased oxidative stress.6Alexandre J Nicco C Chereau C Laurent A Weill B Goldwasser F Batteux F Improvement of the therapeutic index of anticancer drugs by the superoxide dismutase mimic mangafodipir.J Natl Cancer Inst. 2006; 98: 236-244Crossref PubMed Scopus (156) Google Scholar In serum and peritoneal fluid of patients with endometriosis, markers of oxidative stress have been found elevated.7Arumugam K Dip YC Endometriosis and infertility: the role of exogenous lipid peroxides in the peritoneal fluid.Fertil Steril. 1995; 63: 198-199Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 8Oner-Iyidogan Y Kocak H Gurdol F Korkmaz D Buyru F Indices of oxidative stress in eutopic and ectopic endometria of women with endometriosis.Gynecol Obstet Invest. 2004; 57: 214-217Crossref PubMed Scopus (36) Google Scholar, 9Shanti A Santanam N Morales AJ Parthasarathy S Murphy AA Autoantibodies to markers of oxidative stress are elevated in women with endometriosis.Fertil Steril. 1999; 71: 1115-1118Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 10Szczepanska M Kozlik J Skrzypczak J Mikolajczyk M Oxidative stress may be a piece in the endometriosis puzzle.Fertil Steril. 2003; 79: 1288-1293Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar A new role for reactive oxygen species (ROS) as second messenger of cellular proliferation was described. McCubrey et al found that normal cell proliferation correlated with production of endogenous ROS through the activation of growth-related signaling pathways, including the mitogen-activated protein kinase ERK1/2 (extracellular regulated kinase).11McCubrey JA Lahair MM Franklin RA Reactive oxygen species-induced activation of the MAP kinase signaling pathways.Antioxid Redox Signal. 2006; 8: 1775-1789Crossref PubMed Scopus (632) Google Scholar In cancer, modulation of ROS production control tumor cell proliferation,12Laurent A Nicco C Chereau C Goulvestre C Alexandre J Alves A Levy E Goldwasser F Panis Y Soubrane O Weill B Batteux F Controlling tumor growth by modulating endogenous production of reactive oxygen species.Cancer Res. 2005; 65: 948-956PubMed Google Scholar and mutations in mitochondrial DNA resulting in respiratory complex I deficiency have been found to increase endogenous ROS production and enhance metastatic potential of tumor cells.13Ishikawa K Takenaga K Akimoto M Koshikawa N Yamaguchi A Imanishi H Nakada K Honma Y Hayashi J ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis.Science. 2008; 320: 661-664Crossref PubMed Scopus (1065) Google Scholar Endometriosis is considered a benign disease but shares some features with cancer, such as propensity to invasion, unrestrained growth, neoangiogenesis, and distant spreading.14Swiersz LM Role of endometriosis in cancer and tumor development.Ann NY Acad Sci. 2002; 955: 281-295Crossref PubMed Scopus (122) Google Scholar The known correlation between ROS and proliferation of cancer cells, along with the increased production of ROS in response to chronic inflammation in endometriosis, thus suggests a possible role for ROS in the regulation of endometriotic cell proliferation. We thus hypothesized that the development of endometriosis is not solely due to continuous addition of endometrial tissue through menstrual regurgitation in the peritoneum but rather is due to the acquisition of a pro-proliferative phenotype by ectopic endometrial cells through a dysregulation of endogenous ROS production. Endometrium and ovarian endometrioma specimens were obtained from 14 patients with endometriosis undergoing surgical treatment. Control endometrial specimens were obtained from 14 patients without macroscopic endometriosis undergoing laparoscopy for other reasons (tubal infertility, non-endometriotic ovarian cyst, myoma). Written informed consent was obtained from each patient. For each of those 14 patients, a biopsy of eutopic endometrium and of the ovarian endometrioma was performed. Epithelial cell lines and stromal cell lines were derived from biopsies of both eutopic endometrium (endometrial cells) and of ovarian endometrioma (endometriotic cells) from those 14 patients. Fourteen other patients without endometriosis were included in the study as a control group. For each control patient, a biopsy of eutopic endometrium was obtained. For each specimen, epithelial and stromal cells were extracted. Thus, 14 primary epithelial and 14 stromal control cell lines were extracted from biopsies of eutopic endometrium from patients without endometriosis. All of the patients were suffering from chronic inflammation or chronic pelvic pain. C-reactive protein levels, as assessed by the ultrasensitive immunonephelometric method, were within reference ranges for each patient and did not differ from the control group, demonstrating that no patients had acute inflammation at the time of sampling. No differences were observed between the two groups of patients in terms of age, treatment received before surgery, or phase of the menstrual cycle. Patients’ characteristics are summarized in Table 1.Table 1Patient CharacteristicsPatients with endometriosis (n = 14)Control (n = 14)Age, mean ± SEM31.9 ± 1.2232 ± 1.09Cycle n (%) Anovulation9 (64.3)4 (28.6) Proliferative phase4 (28.6)4 (28.6) Secretory phase1 (7.1)6 (42.8)Treatment before surgery n (%) None5 (35.7)10 (64.3) Estroprogestatives2 (14.3)2 (14.3) Progestatives3 (21.45)2 (14.3) Danazol1 (7.1)LHRH agonists3 (21.45)AFS score, mean ± SD3 ± 1C-reactive protein, mean ± SEM2.44 ± 1.011.52 ± 0.42 Open table in a new tab Primary endometrial and endometrioma cell cultures were prepared from biopsies as described.15Hornung D Ryan IP Chao VA Vigne JL Schriock ED Taylor RN Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells.J Clin Endocrinol Metab. 1997; 82: 1621-1628Crossref PubMed Scopus (232) Google Scholar Biopsy specimens were rinsed, minced into small pieces, digested with dispase and collagenase (2 mg/ml, Gibco Invitrogen, Cergy Pontoise, France) for 1 hour at 37°C and separated using serial filtration. Red blood cells were removed by hypotonic lysis (NH4Cl, 0.15 mol/L; KHCO3, 1 mmol/L; Na2 EDTA, 0.1 mmol/L). Debris was removed by 100-μm aperture sieves. Epithelial cells were retained on 40-μm aperture sieves while stromal cells remain in the filtrate. Both cells were plated onto Primaria flasks (Becton Dickinson Labware, Le Pont de Claix, France) and cultured in Dulbecco’s modified Eagle’s medium (Gibco Invitrogen, Cergy Pontoise, France) with 10% fetal calf serum. Purification of stromal and epithelial cells was confirmed by staining with a 1:100 dilution of fluorescein isothiocyanate-labeled anti-cytokeratin and Cy3-labeled anti-vimentin antibodies (Sigma Aldrich, Saint Louis, MO). Fluorescence was analyzed on an Olympus fluorescent microscope (Hamburg, Germany) and images were captured using the CellM Imaging station (Olympus). Both populations were negative for CD3 (T cell), CD45 (leukocyte), and CD11b (monocyte and granulocyte) antibody staining.15Hornung D Ryan IP Chao VA Vigne JL Schriock ED Taylor RN Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells.J Clin Endocrinol Metab. 1997; 82: 1621-1628Crossref PubMed Scopus (232) Google Scholar Cells (104 per well) were seeded in 96-well plates and incubated for 18 hours in triplicate with 6.4 mmol/L N-acetyl-l-cysteine (NAC), 8 mmol/L diethyldithiocarbamate, 40 μmol/L diphenyleneiodonium, 40 μmol/L allopurinol, or culture medium alone. Separately, cells (104 per well) were distributed in triplicates into 96-well plates and, after 18 hours, treated with either 10 μmol/L rotenone or 10 μmol/L antimycin for 30 minutes (all products from Sigma-Aldrich, Saint Louis, MO). Cells were then washed three times in phosphate-buffered saline (PBS) and incubated with 100 μl per well of 5 μmol/L dihydroethidium in PBS for superoxide anion () measurement, or with 100 μl per well of 5 μmol/L 2′,7′-dichlorodihydrofluorescein diacetate in PBS for hydrogen peroxide (H2O2) assay or with 100 μl per well of 5 μmol/L monochlorobimane in PBS for glutathione (GSH) determination. Cellular levels of , H2O2 and GSH were assessed by spectrofluorimetry using a Fusion spectrofluorimeter (Packard Bell, Paris, France). Fluorescence intensity was recorded every hour for 5 hours. Fluorescence excitation/emission maxima were for dihydroethidium, 480/610 nm; for 2′,7′-dichlorodihydrofluorescein diacetate, 507/525 nm; and for monochlorobimane, 380/485 nm.16Servettaz A Guilpain P Goulvestre C Chereau C Hercend C Nicco C Guillevin L Weill B Mouthon L Batteux F Radical oxygen species production induced by advanced oxidation protein products predicts clinical evolution and response to treatment in systemic sclerosis.Ann Rheum Dis. 2007; 66: 1202-1209Crossref PubMed Scopus (79) Google Scholar Fluorescent probes were purchased from Molecular Probes (Eugene, OR). The levels of , H2O2, and GSH were calculated in each sample as follows: reactive oxygen species rate (arbitrary units/min/106 cells) = (fluorescence intensity [arbitrary units] at T 300 minutes − fluorescence intensity [arbitrary units] at T 0/300 minutes/number of adherent cells determined by the crystal violet assay as a measure for membrane integrity. In brief, cells were stained with 0.5% crystal violet and 30% ethanol in PBS for 30 minutes at room temperature. After two washes in PBS, the stained cells were resuspended in 50% methanol, and absorbance was measured at 560 nm on an enzyme-linked immunosorbent assay multiwell reader. Production and metabolism of , H2O2, and GSH were explored in all of the cell lines extracted from the 14 controls and from the 14 endometriotic patients. The superoxide dismutase (SOD) activities of cells were evaluated by the nitroblue tetrazolium reduction technique according to Beauchamp and Fridovich.17Beauchamp C Fridovich I Superoxide dismutase: improved assays and an assay applicable to acrylamide gels.Anal Biochem. 1971; 44: 276-287Crossref PubMed Scopus (9906) Google Scholar The catalase activities of cells were determined by ultraviolet spectroscopy at 240 nm according to Aebi.18Aebi H Catalase in vitro.Methods Enzymol. 1984; 105: 121-126Crossref PubMed Scopus (18681) Google Scholar SOD and catalase measurements were reported to the amount of proteins in each sample (bovine serum albumin microbiuret assay, Pierce, Bezons, France). The enzymatic activities were explored in all of the cell lines extracted from the 14 controls and from the 14 endometriotic patients. Cellular production of H2O2 was assessed, as previously described, after treating cells in 96-well plates for 18 hours with various amounts of the following treatments: RU-486 (1 μmol/L, 10 μmol/L, and 100 μmol/L) and danazol (2.5 μmol/L, 25 μmol/L, and 250 μmol/L) (Sigma Aldrich, Saint Louis, MO). The modulation of in vitro H2O2 production by drugs used in the treatment of endometriosis was explored in all of the cell lines extracted from the 14 controls and from the 14 endometriotic patients. Cells (104 per well) were seeded in 96-well plates and incubated for 48 hours with various amounts of antioxidant and pro-oxidant enzymes or drugs as indicated previously. Cell proliferation was determined by pulsing the cells with [3H]thymidine (1 μCi per well, Amersham, GE HealthCare) during the last 16 to18 hours of culture and measuring the radioactivity incorporated by liquid scintillation counting.16Servettaz A Guilpain P Goulvestre C Chereau C Hercend C Nicco C Guillevin L Weill B Mouthon L Batteux F Radical oxygen species production induced by advanced oxidation protein products predicts clinical evolution and response to treatment in systemic sclerosis.Ann Rheum Dis. 2007; 66: 1202-1209Crossref PubMed Scopus (79) Google Scholar Cell apoptosis was analyzed using Hoechst 33342 staining (Molecular Probes) and a Fusion spectrofluorimeter (Packard Bell). Cells were incubated in 100 μl of PBS with 1 μg/ml Hoechst 33342 for 30 minutes. Fluorescence intensity was recorded at the end of the incubation with a fluorescence excitation/emission maxima of 350/461 nm.16Servettaz A Guilpain P Goulvestre C Chereau C Hercend C Nicco C Guillevin L Weill B Mouthon L Batteux F Radical oxygen species production induced by advanced oxidation protein products predicts clinical evolution and response to treatment in systemic sclerosis.Ann Rheum Dis. 2007; 66: 1202-1209Crossref PubMed Scopus (79) Google Scholar Apoptotic cells were detected by the bright staining of condensed chromatin. The rate of cell proliferation and the viability of cells were determined in all of the cell lines extracted from the 14 controls and from the 14 endometriotic patients. Cells were lysed in ice-cold RIPA buffer (10 mmol/L Tris-HCl, pH 7.5; NaCl, 150 mmol/L; 1% Triton X-100; 0.1% sodium dodecyl sulfate) supplemented with 25 mmol/L sodium fluoride, anti-protease 1%, and 0.5 mmol/L sodium orthovanadate. Equal amounts of protein (30 μg) were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transfer and blocking, nitrocellulose membrane was incubated overnight at 4°C with a 1:200 dilution of a rabbit anti-human ERK 1/2 IgG and a 1:200 dilution of a rabbit anti-human pERK 1/2 IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Specific proteins were detected using a 1:1000 dilution of a horseradish peroxidase-conjugated goat anti-rabbit IgG and visualized by an enhanced chemiluminescence system (Pierce Perbio Sciences, Brebières, France).19Song JS Kang CM Yoo MB Kim SJ Yoon HK Kim YK Kim KH Moon HS Park SH Nitric oxide induces MUC5AC mucin in respiratory epithelial cells through PKC and ERK dependent pathways.Respir Res. 2007; 8: 28-40Crossref PubMed Scopus (38) Google Scholar ERK and pERK were explored in all of the cell lines extracted from the 14 controls and from the 14 endometriotic patients. Twenty-eight female nude mice (Swiss nu/nu, Charles River Laboratories, L’Arbresle, France) 8 weeks of age were used in this study. Animals received humane care in compliance with institutional guidelines. Mice were anesthetized with intraperitoneal injections of 500 μl of Avertine (2,2-tribromoethanol, 0.04 mol/L; 2 methyl-2-butanol, 2.5%). An incision was made on the ventral midline and one 5-mm2 fragment of human endometrioma was sutured to the parietal peritoneum with 7/0 Proline. The cutis was sutured with a 6/0 nylon thread.20Bruner-Tran KL Webster-Clair D Osteen KG Experimental endometriosis: the nude mouse as a xenographic host.Ann NY Acad Sci. 2002; 955: 328-342Crossref PubMed Scopus (64) Google Scholar On day 14 after implantation, mice were operated to confirm the viability of the implant. Treatments started on day 14: the first group received subcutaneous injections of RU-486 (50 mg/kg in 200 μl of sesame oil, three times a week); the second group received subcutaneous injections of danazol (25 mg/kg in 200 μl of sesame oil, three times a week)21Ghoshal K Majumder S Zhu Q Hunzeker J Datta J Shah M Sheridan JF Jacob ST Influenza virus infection induces metallothionein gene expression in the mouse liver and lung by overlapping but distinct molecular mechanisms.Mol Cell Biol. 2001; 21: 8301-8317Crossref PubMed Scopus (56) Google Scholar, 22Kusakabe K Morishima S Nakamuta N Li ZL Otsuki Y Effect of danazol on NK cells and cytokines in the mouse uterus.J Reprod Dev. 2007; 53: 87-94Crossref PubMed Scopus (7) Google Scholar; and the third group received intraperitoneal injections of NAC (60 mg/mouse, in 300 μl of PBS, three times a week) according to the protocol of Marian et al23Marian AJ Senthil V Chen SN Lombardi R Antifibrotic effects of antioxidant N-acetylcysteine in a mouse model of human hypertrophic cardiomyopathy mutation.J Am Coll Cardiol. 2006; 47: 827-834Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar slightly modified. Indeed, to minimize the infectious risks in nude mice, the animals were injected with twice the amount of drug every other day instead of a single amount of drug every day. Moreover, to limit the stress of mice injected intraperitoneally with NAC, we decided not to weigh NAC-injected mice on each day of injection, but to use a medium dose of 60 mg per injection three times a week. Using this regimen, the NAC-treated mice received a cumulative dose of 720 mg. This dose is very similar to the dose that the mice treated with NAC 1000 mg/kg/day received according to the original protocol. Mice of 20 to 22 g were implanted with endometriotic biopsies at day 0. Treatment with NAC started 2 weeks later. The control group received intraperitoneal injections of 300 μl of PBS or subcutaneous injections of 200 μl of sesame oil three times a week. After 4 weeks of treatment, animals were sacrificed by cervical dislocation. Implants were extracted for histological analysis. Slides were stained with hematoxylin-eosin. Pathology scores were derived as follows: 0 (receded lesion with stromal fibrosis, hemosiderin and absence of glandular structure) to 3 (active lesion with fresh blood, profuse stromal cellular infiltration and developed glandular organization). Scoring was performed by three different scientists unaware of the treatments received in each group. All of the results of in vitro studies are the means of independent triplicate experiments for each cellular population of each patient. Statistical analysis was performed with StatView 3.0 software (SAS Institute Inc., Cary, NC), according to the practice of the biostatistics department of our institution.24Wang M Jeng KC Ping LI Exogenous cytokine modulation or neutralization of interleukin-10 enhance survival in lipopolysaccharide-hyporesponsive C3H/HeJ mice with Klebsiella infection.Immunology. 1999; 98: 90-97Crossref PubMed Scopus (27) Google Scholar Means were compared by Student’s t-test. In all figures, error bars represent the SEM. A level of P < 0.05 was accepted as significant (*P < 0.05, **P < 0.01, ***P < 0.001). Fourteen patients with ovarian endometrioma were included in the study. For each of those patients, a biopsy of eutopic endometrium and of the ovarian endometrioma was performed. Fourteen patients without endometriosis were included in the study as a control group. For each control patient, a biopsy of eutopic endometrium was obtained. For each specimen, epithelial and stromal cells were extracted. Thus, 14 primary epithelial and stromal cell lines were extracted from biopsies of eutopic endometrium from patients without endometriosis (control cells), and from biopsies of both eutopic endometrium (endometrial cells) and ovarian endometrioma (endometriotic cells) from 14 patients with endometriosis. Examples of stromal and epithelial cell lines derived from the biopsies of patients are shown in Figure 1, A–F. The production of O2− was increased by 39% in stromal endometriotic cells from patients (P < 0.05) and by 35% in stromal endometrial cells (P<0.05) compared with stromal control cells. Production of O2− was not significantly different between the various epithelial cell lines tested (Figure 2A). The treatment received before surgery did not affect the results gained in endometriotic cell lines. O2− is detoxified by SOD to form H2O2. SOD activity was found to be threefold higher in stromal endometriotic cells (P < 0.01) and 2.25-fold higher in stromal endometrial cells from patients (P < 0.05) than in stromal control cells (Figure 2B). SOD activity was 3.4-fold higher in epithelial endometriotic cells (P < 0.001) and twofold higher in epithelial endometrial cells from patients (P < 0.05), than in epithelial control cells (Figure 2B). Treating the cells with diethyldithiocarbamate, a specific inhibitor of SOD activity, increased O2− production by 60% in stromal endometriotic cells (P < 0.05), 50% in stromal endometrial cells (P < 0.05), 70% in epithelial endometriotic cells (P < 0.01), and 40% in epithelial endometrial cells (P < 0.05), compared with untreated cells (Figure 2C). Thus, the increased production of superoxide anions in endometriotic cells is associated with an increase in SOD activity as a compensatory feedback mechanism to control increased oxidative stress. Inhibiting SOD blocked this adaptive mechanism and induced an oxidative burst. Superoxide anion is generated by either the mitochondrial respiratory chain or the cytosolic enzymes NADPH oxidase and xanthene oxidase. Several inhibitors of mitochondrial respiratory complexes and of cytosolic enzymes were used to explore the origin of superoxide anions in endometriotic and endometrial cell lines. Rotenone and antimycin blocked the mitochondrial respiratory chain complexes I and III, respectively. This blockage led to the release of electrons from the respiratory chain, which combined with molecular oxygen to produce superoxide anions. Therefore, if a disturbance of complex I or III is responsible for the production of superoxide anions, the treatment with rotenone or antimycin will increase this dysfunction and the release of superoxide anions. By contrast, if superoxide anions originated from the cytosolic enzyme NAD(P)H oxidase, their production would be abrogated by treatment with the selective inhibitor of NAD(P)H oxidase, diphenyleneiodonium. Alternatively, if superoxide anions originated from the cytosolic enzyme xanthene oxidase, their production would be decreased by treatment with the selective inhibitor of xanthene oxidase, allopurinol.25Laurent A Nicco C Tran Van Nhieu J Borderie D Chereau C Conti F Jaffray P Soubrane O Calmus Y Weill B Batteux F Pivotal role of superoxide anion and beneficial effect of antioxidant molecules in murine steatohepatitis.Hepatology. 2004; 39: 1277-1285Crossref PubMed Scopus (112) Google Scholar In stromal endometriotic cells, O2− production was significantly decreased by diphenyleneiodonium (P < 0.05) and by allopurinol (P < 0.05). In epithelial endometriotic cells, O2− production was significantly increased by the inhibition of the mitochondrial respiratory chain complex I by rotenone (P < 0.01) and complex III by antimycin (P < 0.001) (Figure 2D). Thus, these data show that in stromal endometriotic cells, O2− is mostly produced by cytosolic enzymes as in normal cell lines, whereas in epithelial endometriotic cells, it is produced by mitochondrial complex I and III as in tumor cells (Figure 2D). Hydrogen peroxide is a byproduct of O2− detoxification by SOD. H2O2 production was 2.5-fold higher in stromal endometriotic cells than in stromal control cells (P < 0.05). In epithelial endometriotic cells, production of H2O2 was 7.75-fold higher than in epithelial control cells (P < 0.001) and 3.1-fold higher than in epithelial endometrial cells (P < 0.001) (Figure 3A). The treatment received before surgery did not affect the results gained in endometriotic cell lines. The detoxification of H2O2 is achieved through two different enzymatic systems, GSH peroxidase and catalase. Glutathione peroxidase is an enzyme that uses reduced glutathione to convert H2O2 into water whereas catalase can do so directly. We found that catalase activity was 2.7-fold lower in stromal endometriotic cells than in stromal control cells (P < 0.01) and 1.8-fold lower than in stromal endometrial cells (P < 0.01) (Figure 3B). Catalase activity was threefold lower in epithelial endometriotic cells (P < 0.01) and 1.5-fold lower in epithelial endometrial cells (P < 0.05) compared with epithelial control cells. The level of GSH was 1.6-fold higher in stromal endometriotic cells than in stromal control cells (P < 0.01). In epithelial endometriotic cells, the level of GSH was 1.2-fold higher when compared with epithelial control cells (P < 0.05) (Figure 3C). These results show that the high level of H2O2 in endometriotic cells results from both an increase in its production by the co
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