Endometrial hyperplasia complex is gynecological disorder characterized by morphological irregularities of glands shape and size. Antioxidant enzymes (AOE), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), have an essential role in preventing oxidative damage in cell caused by reactive oxygen species (ROS). In this study, we examined the AO status in hyperplastic tissue of patients in menstrual cycle (follicular and luteal phase) and in postmenopause, as well as the relationship between sex hormones and AO parameters. The phase-related activity of GPx and GR in examined patients was significantly different than in healthy women. A significant negative correlation between FSH/LH level and GPx activiy was observed. Endometrial hyperplasias are considered as precancerous lesions and are treated either conservatively or surgicaly, and also by radiation therapy. Since the effects of these therapies are associated with AO and hormonal changes, our results may contribute to the prediction of potential therapeutic efficacy and to selection of the most effective treatment for hyperplasia complex.3rd International Conference on Radiation and Applications in Various Fields of Research (RAD), Jun 08-12, 2015, Budva, Montenegr

Gavrilović, Ljubica

Pajović, Snežana B.

Pavlović, Ivan

Pejić, Snežana

Popović, Nataša M.

Stojiljković, Vesna

Todorović, Ana

English

Repository of the Vinča Nuclear Institute (VinaR)

PUBLISHER: RAD Association, Niš, Serbia www.rad-association.org  FOR THE PUBLISHER: Prof. Dr. Goran Ristić  EDITOR: Prof. Dr. Goran Ristić  COVER DESIGN: Vladan Nikolić, M.Sc.   TECHNICAL EDITING: Sasa Trenčić and Vladan Nikolić  PROOF-READING: Saša Trenčić, MA and Mila Aleksov, BA   ISBN: 978-86-80300-01-6     CIP - Каталогизација у публикацији -  Народна библиотека Србије, Београд    539.16(082)(0.034.2)    INTERNATIONAL Conference on Radiation and Applications in Various Fields  of Research (3rd ; 2015 ; Budva) Proceedings [Elektronski izvor] / Third International Conference on  Radiation and Applications in Various Fields of Research, RAD 2015, June  8-12, 2015, Budva, Montenegro ; [editor Goran Ristić]. - Niš : RAD  Association, 2015 (Niš : RAD Association). - 1 elektronski optički disk  (CD-ROM) ; 12 cm    Sistemski zahtevi: Nisu navedeni. - Nasl. sa naslovne strane dokumenta. -  Tiraž 400. - Bibliografija uz svaki rad.    ISBN 978-86-80300-01-6    a) Јонизујуће зрачење - Дозиметрија - Зборници  COBISS.SR-ID 215655436 ANTIOXIDANT ENZYMES IN WOMEN WITH HYPERPLASIA COMPLEX:  RELATION WITH SEX HORMONES Snežana Pejić, Ana Todorović, Vesna Stojiljković, Ljubica Gavrilović, Nataša Popović, Ivan Pavlović, Snežana Pajović Laboratory of Molecular Biology and Endocrinology, "Vinča" Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia Abstract. Endometrial hyperplasia complex is gynecological disorder characterized by morphological irregularities of glands shape and size. Antioxidant enzymes (AOE), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), have an essential role in preventing oxidative damage in cell caused by reactive oxygen species (ROS). In this study, we examined the AO status in hyperplastic tissue of patients in menstrual cycle (follicular and luteal phase) and in postmenopause, as well as the relationship between sex hormones and AO parameters. The phase-related activity of GPx and GR in examined patients was significantly different than in healthy women. A significant negative correlation between FSH/LH level and GPx activiy was observed. Endometrial hyperplasias are considered as precancerous lesions and are treated either conservatively or surgicaly, and also by radiation therapy. Since the effects of these therapies are associated with AO and hormonal changes, our results may contribute to the prediction of potential therapeutic efficacy and to selection of the most effective treatment for hyperplasia complex.  Key words: endometrial hyperplasia, antioxidant enzymes, steroid hormones    Introduction According to WHO (World Health Organization) classification, hyperplasia complex is a type of endometrial hyperplasia and could be with atypical cytology or without atypia (1). The main features of this condition, as in the other types of endometrial hyperplasia, are morphological irregularities in endometrium caused by changes in the glands shape and size. This leads to rise in the gland to stroma ratio when compared with normal endometrium (2). Beside the age, obesity, hormone replacement therapy and diabetes are among the risk factors significant for hyperplasia development (3). Endometrial tissue is under a strong influence of sexual hormones and endometrium exposure to an excessive amount of estrogen unopposed by progesterone leads to abnormal proliferation (4). Therefore, hormones are considered as one of the key factors in the development of endometrial hyperplasia and endometrial cancer. Reactive oxygen species (ROS) are produced during cell physiological activity. When ROS production overcomes the antioxidant capacity of the cell, the result is oxidative stress, which may lead to different pathological conditions including cancer. Antioxidant enzymes (AOE) have an essential role in preventing oxidative damage in cell caused by reactive oxygen species. Superoxide dismutases (SODs) remove superoxide anion (O2.-), catalyzing its reduction to hydrogen peroxide (H2O2). Hydrogen peroxide becomes a substrate for two enzymes, catalase (CAT) and glutathione peroxidase (GPx) that reduce H2O2 into water (5). For its catalytic activity, GPx requires glutathione (GSH) as a hydrogen donor, coupling its oxidation to GSSG with reduction of H2O2. Glutathione itself acts as antioxidant by scavenging reactive oxygen species. Glutathione reductase (GR) converts GSSG into GSH (6). The AO status and hormone influence were studied during the menstrual cycle and postmenopause in healthy women and in some gynaecological disorders. We have shown that AO enzymes activity and lipid hydroperoxide (LOOH) level in patients with endometrial polyps are influenced by the changes in sex hormones during the menstrual cycle and in menopause (7). In this study, we examined the AO status in menstrual cycle and postmenopause of women with endometrial hyperplasia complex and a relationship between sex hormones and AO parameters.  Methods Subjects. The material used in this study consisted of 31 endometrial tissue specimens of women admitted to the Department of Gynecology and Obstetrics for gynecological evaluation within routine checkups or for abnormal uterine bleeding. Study was conducted prospectively and it was approved by the Human Studies Ethics Committee of the Clinical Center. The protocol was consistent with the World Medical Association Declaration of Helsinki. None of them had undergone hormone therapy or any other medical treatment in the last 6 months. Women were divided in three groups: women in proliferative (follicular phase, F), in secretory (luteal phase, L), and women in postmenopause (PM). Endometrial samples were washed in saline solution and homogenized in phosphate buffer containing 0.05M KH2PO4 and 1 mM EDTA, pH 7.8 and frozen at -70ºC for 20 h in order to disrupt cell membranes. For SOD assay, thawed homogenates were vortexed 1 min and centrifuged at 8600 g, for 20 min at 4ºC. According to manufacturer’s recommendation, after addition of ethanol/chloroform extraction reagent (62.5/37.5 vol/vol) to completely remove hemoglobin interference, samples were centrifuged at 6000 g for 20 min, at 4ºC. Upper aqueous layer was collected and kept at -70ºC until assay. The enzyme activities and lipid hydroperoxide (LOOH) concentration were monitored spectrophotometrically using Oxis Bioxytech® Assays (Oxis International, Inc., Portland, OR, USA).  Assay of SOD activity. The method is based on SOD-mediated increase of autoxidation of 5,6,6a11b-tetrahydro-3,9,10-tryhydroxybenzo[c]fluorene in aqueous alkaline solution to yield a chromophore with maximum absorbance at 525 nm. The SOD activity is determined from the ratio of the autoxidation rates in the presence (Vs) and in the absence (Vc) of SOD. One SOD activity unit is defined as the activity that doubles the autoxidation rate of the control blank.    Assay of CAT activity. CAT activity was determined by the method of Beutler (8). The reaction is based on the rate of H2O2 degradation by catalase contained in the examined samples. The reaction was performed in an incubation mixture containing 1M Tris-HCl, 5mM EDTA, pH 8.0, and monitored at 230 nm. One unit of CAT activity is defined as 1 µmol of H2O2 decomposed per minute under the assay conditions.   Assay of GPX activity. GPx activity was assessed using the principle that oxidized glutathione (GSSG) produced upon reduction of an organic peroxide by GPx, is immediately recycled to its reduced form (GSH) with concomitant oxidation of NADPH to NADP+. The oxidation of NADPH was monitored as a decrease in absorbance at 340 nm. One GPx unit is defined as 1 µmol of NADH oxidized per minute under the assay conditions.   Assay of GR activity. Assay is based on the oxidation of NADPH to NADP+ during the reduction of oxidized glutathione (GSSG), catalyzed by a limiting concentration of glutathione reductase. The oxidation of NADPH was monitored as a decrease in absorbance at 340 nm. One GR unit is defined as 1 µmol of NADH oxidized per minute under the assay conditions.   Lipid hydroperoxides. Concentration of LOOH measurement is based on the oxidation of ferrous (Fe2+) ions to ferric (Fe3+) ions by hydroperoxides under acidic conditions. Ferric ions then bind with the indicator dye, xylenol orange, and form a colored complex. The absorbance of the complex was measured at 560 nm. Since hydrogen peroxide content in many biological samples is much higher than that of other hydroperoxides, samples were pretreated with catalase to decompose the existing H2O2 and eliminate the interference.   The specific enzyme activities were expressed as U or mU/mg protein. LOOH concentration was expressed as nmol/mg protein. Protein concentration was determined by the method of Lowry et al. (9) and expressed as mg/ml.       Serum hormone levels were analysed in the hormone analysis laboratory. Estradiol (E) and progesterone (P) were determined by the RIA-CT technique used for quantitative determination of E or P in human serum or plasma. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were determined by the IRMA FSH and LH test, which determines the quantity of immune-radiometric human FSH and LH in serum.       Statistics. Statistical analysis was carried out by use of the Kruskal-Wallis test and the Dunn’s post hoc test, which considered the unequal and small sample sizes we used in this study. A linear regression model was used to evaluate associations between hormonal and antioxidant variables. Before plotting the data in the regression study, the normality test on the variables was performed and the values of estradiol and progesterone were log-transformed. Two-tailed p values are given throughout. All data were analyzed using GraphPad Prism software.  Results Significant changes were observed for all hormone concentrations, FSH (H=5.99, p<0.05), LH (H=7.11, p<0.05), estradiol (H=8.10, p<0.05), and progesterone (H=6.42, p<0.05), (Table 1). We also found significant phase-related changes of GPx (H=7.84, p<0.05) and 468GR activity (H=8.10, p<0.05). Both enzymes had similar activity pattern, which was higher in follicular phase compared to luteal phase and to postmenopause, (p<0.05). The phase-related activity of SOD and CAT as well as the LOOH level did not show any statistical difference (Table 2). The linear regression analysis of individual hormonal variables against antioxidant parameters in endometrium showed a significant negative correlation between FSH/LH concentrations and GPx activity (r= -0.36; r= -0.47, p<0.05, respectively).  Table 1. Hormone levels during follicular phase, luteal phase and in postmenopause (Data are expressed as mean ± SEM; * p<0.05).  Table 2. LOOH concentrations and AO enzyme activities in follicular phase (F), luteal phase (L) and postmenopause (PM) in hyperplasia complex tissue. Data are shown as mean ± SEM. P values refer to the results of the Dunn test.      Discussion Previous studies of AO enzymes in endometrium pointed to hormone-dependent expression and activity. Ota et al. (10) have shown the phase-dependent changes of  both forms of SODs (copper, zinc SOD and manganese SOD) in glandular and surface epithelia of healthy fertile women during the menstrual cycle. Specifically, their expression was lowest during the early and midproliferative phases, then gradually increased and was most pronounced in the early and mid-secretory phases. In women with endometriosis and adenomyosis, the expression of both SODs was constantly elevated. Some data also indicated that SOD activity was not different in postmenopausal women compared to fertile ones (11). Our recent findings in women with endometrial polyp showed the opposite pattern  of LOOH concentration and SOD activity in polyp tissue than in women with hyperplasia complex. Both parameters were higher in the proliferative phase compared to the secretory phase or to the women in postmenopause (7). The results of this study showed that women with endometrial hyperplasia complex had similar SOD activity pattern like the healthy ones. Although it was not statistically significant, SOD activity slightly increased in luteal (secretory) phase.  Regarding GPx, the studies shown that in normal human endometrium the amount of this enzyme was low in the early proliferative phase, gradually increased and reached a maximum in the late proliferative/early secretory phases, and decreased thereafter (12). In this study, however, the GPx activity in hyperplastic tissue had the opposite pattern than in healthy women. The same variation of activity was observed for GR. Ohwada et al. (13) also found that GPx activity is significantly higher in endometrial cancer tissue with atypical or mixed atypical adenomatous hyperplasia than in endometrial cancer without hyperplasia, pointing to the role of AO status and ROS in hyperplastic changes that may lead to endometrial cancer.  In this study we recorded a negative correlation between GPx and gonadotropins in women with endometrial hyperplasia complex, which may indicate the important role of these hormones in hyperplastic changes. Our previous research also showed a modulatory effect of gonadotropins on AO enzymes in women with endometrial polyp (7). Endometrial hyperplasias are generally considered as precancerous lesions and are treated either conservatively or surgicaly, and also by radiation therapy (14). The regression of hyperplastic to normal endometrium is the main purpose of any conservative treatment which is based on the administration of agents, like progestagens (15)   F (n=10)  L (n=10) PM (n=11)  FSH  (U/L)*   5.97±1.42   18.06±4.87   25.59±5.62   LH  (U/L)*   0.60±0.30   6.07±1.70   12.02±4.33   Estradiol (pg/ml)*   69.47±29.21   73.07±21.81   21.27±3.75   Progesterone (nmol/l)*   1.10±0.10   5.97±0.68   4.83±0.84   F L PM SOD (U/mg protein) 0.47 ± 0.05 0.54 ± 0.07 0.66 ± 0.07  CAT  70.18 ± 10.54  77.34 ± 10.73 67.52 ± 14.80 (U/mg protein)  GPx (mU/mg protein)  37.44 ± 3.27*  21.48 ± 1.81  19.84 ± 1.60  GR (mU/mg protein)  5.96 ± 0.47* 3.41 ± 0.65 3.313 ± 0.42 LOOH (nm/mg protein) 0.32 ± 0.04 0.34 ± 0.03 0.35 ± 0.02 469or gonadotropin-releasing hormone analogues (GnRHa) (16). Since the effects of these therapies are associated with AO and hormonal changes, our results may contribute to the prediction of potential therapeutic efficacy and to selection of the most effective treatment for hyperplasia complex. Acknowledgement This work was financially supported by the Ministry of Education, Science and Technological Development (Grants 41027, 41022, 173041). Reference  1. M.M. Hannemann, H.M. Alexander, N. J. Cope, N. Acheson and A.Phillips, “ Endometrial hyperplasia: a clinician's review“,  Obstet. Gynaecol. Reprod. Med.,  vol.20, pp. 116-120, 2010. 2. L.C. Horn, A. Meinel, R. Handzel and J. Einenkel, “Histopathology of endometrial hyperplasia and endometrial carcinoma: an update“, Ann. Diagn. Pathol., vol. 11, pp. 297-311, 2007. 3. F. Linkov, R. Edwards, J. Balk, Z. Yurkovetsky, B. Stadterman, A. Lokshin and E. Taioli, “Endometrial hyperplasia, endometrial cancer and prevention: Gaps in existing research of modifiable risk factors“, Eur. J. Cancer Prev., vol. 44, pp. 1632-1644, 2008. 4. M.E. Sherman, “ Theories of endometrial carcinogenesis: a multidisciplinary approach“, Mod. Pathol., vol. 13. pp. 295-308, 2000. 5. T. Rahman, I. Hosen, M.T. Islam and H.U. Shekhar, “ Oxidative stress and human health“, Adv. Biosci. Biotechnol., vol. 3, p. 997, 2012. 6. G. Wu, Y. Z. Fang, S. Yang, J.R. Lupton and N.D. Turner, “ Glutathione metabolism and its implications for health“, J. Nutr., vol.134, pp.489-492, 2004. 7. S.A. Pejic, J.D. Kasapovic, A.U. Todorovic, V.R. Stojiljkovic, L.V. Gavrilovic, N.M. Popovic and S.B. Pajovic, “ Antioxidant enzymes in women with endometrial polyps: relation with sex hormones“, Eur. J. Obstet. Gynecol. Reprod. Biol., vol. 170(1), pp. 241-246, 2013. 8. E. Beutler, “Catalase“, in   Red Cell Metabolism, a Manual of Biohemical Methods 3rd ed., E. Beutler, ed., New York: Grune and Stratton, 1982, pp. 105-106. 9. O.H. Lowry, N.J. Rosenbrough, A.L. Farr and R.J. Randall, “ Protein measurement with the Folin phenol reagent“, J. Biol. Chem., vol. 193, pp. 265-275, 1951. 10. H. Ota, S. Igarashi, J. Hatazawa and T. Tanaka, “ Immunohistochemical assessment of superoxide dismutase expression in the endometrium in endometriosis and adenomyosis“, Fertil. Steril., vol.72(1), pp.129-134, 1999. 11. F. Gürdöl, Y. Öner-Iyidoĝan, Ö. Yalçin, S. Genç and F. Buyru, “Changes in enzymatic antioxidant defense system in blood and endometrial tissues of women after menopause“, Res. Commun. Mol. Pathol. Pharmacol., vol. 97, pp. 38-46, 1997. 12. H. Ota, S. Igarashi, N. Kato and T. Tanaka, “ Aberrant expression of glutathione peroxidase in eutopic and ectopic endometrium in endometriosis and adenomyosis“, Fertil. Steril., vol. 74, pp. 313-318, 2000. 13. M. Ohwada, M. Suzuki, I. Sato, H. Tsukamoto and K. Watanabe, “ Glutathione peroxidase activity in endometrium: effects of sex hormones and cancer“, Gynecol. Oncol., vol. 60, pp. 277-282, 1996. 14. G.S. Silverberg, “Problems in the Differential Diagnosis of Endometrial Hyperplasia and Carcinoma“, Mod. Pathol., vol. 13 (3), pp. 309-327, 2000. 15. I.D. Gallos, M. Shehmar, S. Thangaratinam. T.K. Papapostolou, A. Coomarasamy, J.K. Gupta, “Oral progestogens vs levonorgestrel-releasing intrauterine system for endometrial hyperplasia: a systematic review and metaanalysis“, Am. J. Obstet. Gynecol., vol. 203 (6), pp. 547 e1-10, 2010. 16. G. Grimbizis, T. Tsalikis, V. Tzioufa, M. Kasapis and S. Manatalenakis, ” Regression of endometrial hyperplasia after treatment with the gonadotropin-releasing hormone analogue triptorelin: a prospective study“, Hum. Reprod., vol. 14. pp. 479-484, 1999.  

Antioxidant Enzymes in Women with Hyperplasia Complex: Relation with Sex Hormones

VinaR - Repository of the Vinča Institute of Nuclear Sciences


PUBLISHER: RAD Association, Niš, Serbia 
www.rad-association.org 
 
FOR THE PUBLISHER: Prof. Dr. Goran Ristić 
 
EDITOR: Prof. Dr. Goran Ristić 
 
COVER DESIGN: Vladan Nikolić, M.Sc.  
 
TECHNICAL EDITING: Sasa Trenčić and Vladan Nikolić 
 
PROOF-READING: Saša Trenčić, MA and Mila Aleksov, BA 
 
 
ISBN: 978-86-80300-01-6 
 
   
CIP - Каталогизација у публикацији -  
Народна библиотека Србије, Београд 
   
539.16(082)(0.034.2) 
   
INTERNATIONAL Conference on Radiation and Applications in Various Fields  
of Research (3rd ; 2015 ; Budva) 
Proceedings [Elektronski izvor] / Third International Conference on  
Radiation and Applications in Various Fields of Research, RAD 2015, June  
8-12, 2015, Budva, Montenegro ; [editor Goran Ristić]. - Niš : RAD  
Association, 2015 (Niš : RAD Association). - 1 elektronski optički disk  
(CD-ROM) ; 12 cm 
   
Sistemski zahtevi: Nisu navedeni. - Nasl. sa naslovne strane dokumenta. -  
Tiraž 400. - Bibliografija uz svaki rad. 
   
ISBN 978-86-80300-01-6 
   
a) Јонизујуће зрачење - Дозиметрија - Зборници 
 
COBISS.SR-ID 215655436 
ANTIOXIDANT ENZYMES IN WOMEN WITH HYPERPLASIA COMPLEX:  
RELATION WITH SEX HORMONES 
Snežana Pejić, Ana Todorović, Vesna Stojiljković, Ljubica Gavrilović, Nataša Popović, Ivan 
Pavlović, Snežana Pajović 
Laboratory of Molecular Biology and Endocrinology, "Vinča" Institute of Nuclear Sciences, University of Belgrade, 
Belgrade, Serbia 
Abstract. Endometrial hyperplasia complex is gynecological disorder characterized by morphological irregularities 
of glands shape and size. Antioxidant enzymes (AOE), superoxide dismutase (SOD), catalase (CAT), glutathione 
peroxidase (GPx) and glutathione reductase (GR), have an essential role in preventing oxidative damage in cell caused 
by reactive oxygen species (ROS). In this study, we examined the AO status in hyperplastic tissue of patients in 
menstrual cycle (follicular and luteal phase) and in postmenopause, as well as the relationship between sex hormones 
and AO parameters. The phase-related activity of GPx and GR in examined patients was significantly different than in 
healthy women. A significant negative correlation between FSH/LH level and GPx activiy was observed. Endometrial 
hyperplasias are considered as precancerous lesions and are treated either conservatively or surgicaly, and also by 
radiation therapy. Since the effects of these therapies are associated with AO and hormonal changes, our results may 
contribute to the prediction of potential therapeutic efficacy and to selection of the most effective treatment for 
hyperplasia complex. 
 
Key words: endometrial hyperplasia, antioxidant enzymes, steroid hormones 
 
 
 
Introduction 
According to WHO (World Health 
Organization) classification, hyperplasia 
complex is a type of endometrial hyperplasia and 
could be with atypical cytology or without atypia 
(1). The main features of this condition, as in the 
other types of endometrial hyperplasia, are 
morphological irregularities in endometrium 
caused by changes in the glands shape and size. 
This leads to rise in the gland to stroma ratio 
when compared with normal endometrium (2). 
Beside the age, obesity, hormone replacement 
therapy and diabetes are among the risk factors 
significant for hyperplasia development (3). 
Endometrial tissue is under a strong influence of 
sexual hormones and endometrium exposure to 
an excessive amount of estrogen unopposed by 
progesterone leads to abnormal proliferation (4). 
Therefore, hormones are considered as one of 
the key factors in the development of 
endometrial hyperplasia and endometrial cancer. 
Reactive oxygen species (ROS) are produced 
during cell physiological activity. When ROS 
production overcomes the antioxidant capacity 
of the cell, the result is oxidative stress, which 
may lead to different pathological conditions 
including cancer. Antioxidant enzymes (AOE) 
have an essential role in preventing oxidative 
damage in cell caused by reactive oxygen species. 
Superoxide dismutases (SODs) remove 
superoxide anion (O2.-), catalyzing its reduction 
to hydrogen peroxide (H2O2). Hydrogen 
peroxide becomes a substrate for two enzymes, 
catalase (CAT) and glutathione peroxidase (GPx) 
that reduce H2O2 into water (5). For its catalytic 
activity, GPx requires glutathione (GSH) as a 
hydrogen donor, coupling its oxidation to GSSG 
with reduction of H2O2. Glutathione itself acts as 
antioxidant by scavenging reactive oxygen 
species. Glutathione reductase (GR) converts 
GSSG into GSH (6). 
The AO status and hormone influence were 
studied during the menstrual cycle and 
postmenopause in healthy women and in some 
gynaecological disorders. We have shown that 
AO enzymes activity and lipid hydroperoxide 
(LOOH) level in patients with endometrial 
polyps are influenced by the changes in sex 
hormones during the menstrual cycle and in 
menopause (7). In this study, we examined the 
AO status in menstrual cycle and 
postmenopause of women with endometrial 
hyperplasia complex and a relationship between 
sex hormones and AO parameters. 
 
Methods 
Subjects. The material used in this study 
consisted of 31 endometrial tissue specimens of 
women admitted to the Department of 
Gynecology and Obstetrics for gynecological 
evaluation within routine checkups or for 
abnormal uterine bleeding. Study was conducted 
prospectively and it was approved by the Human 
Studies Ethics Committee of the Clinical Center. 
The protocol was consistent with the World 
Medical Association Declaration of Helsinki. 
None of them had undergone hormone therapy 
or any other medical treatment in the last 6 
months. Women were divided in three groups: 
women in proliferative (follicular phase, F), in 
secretory (luteal phase, L), and women in 
postmenopause (PM). 
Endometrial samples were washed in saline 
solution and homogenized in phosphate buffer 
containing 0.05M KH2PO4 and 1 mM EDTA, pH 
7.8 and frozen at -70ºC for 20 h in order to 
disrupt cell membranes. For SOD assay, thawed 
homogenates were vortexed 1 min and 
centrifuged at 8600 g, for 20 min at 4ºC. 
According to manufacturer’s recommendation, 
after addition of ethanol/chloroform extraction 
reagent (62.5/37.5 vol/vol) to completely 
remove hemoglobin interference, samples were 
centrifuged at 6000 g for 20 min, at 4ºC. Upper 
aqueous layer was collected and kept at -70ºC 
until assay. The enzyme activities and lipid 
hydroperoxide (LOOH) concentration were 
monitored spectrophotometrically using Oxis 
Bioxytech® Assays (Oxis International, Inc., 
Portland, OR, USA).  
Assay of SOD activity. The method is based 
on SOD-mediated increase of autoxidation of 
5,6,6a11b-tetrahydro-3,9,10-
tryhydroxybenzo[c]fluorene in aqueous alkaline 
solution to yield a chromophore with maximum 
absorbance at 525 nm. The SOD activity is 
determined from the ratio of the autoxidation 
rates in the presence (Vs) and in the absence 
(Vc) of SOD. One SOD activity unit is defined as 
the activity that doubles the autoxidation rate of 
the control blank.    
Assay of CAT activity. CAT activity was 
determined by the method of Beutler (8). The 
reaction is based on the rate of H2O2 degradation 
by catalase contained in the examined samples. 
The reaction was performed in an incubation 
mixture containing 1M Tris-HCl, 5mM EDTA, 
pH 8.0, and monitored at 230 nm. One unit of 
CAT activity is defined as 1 µmol of H2O2 
decomposed per minute under the assay 
conditions.   
Assay of GPX activity. GPx activity was 
assessed using the principle that oxidized 
glutathione (GSSG) produced upon reduction of 
an organic peroxide by GPx, is immediately 
recycled to its reduced form (GSH) with 
concomitant oxidation of NADPH to NADP+. 
The oxidation of NADPH was monitored as a 
decrease in absorbance at 340 nm. One GPx unit 
is defined as 1 µmol of NADH oxidized per 
minute under the assay conditions.   
Assay of GR activity. Assay is based on the 
oxidation of NADPH to NADP+ during the 
reduction of oxidized glutathione (GSSG), 
catalyzed by a limiting concentration of 
glutathione reductase. The oxidation of NADPH 
was monitored as a decrease in absorbance at 
340 nm. One GR unit is defined as 1 µmol of 
NADH oxidized per minute under the assay 
conditions.   
Lipid hydroperoxides. Concentration of 
LOOH measurement is based on the oxidation of 
ferrous (Fe2+) ions to ferric (Fe3+) ions by 
hydroperoxides under acidic conditions. Ferric 
ions then bind with the indicator dye, xylenol 
orange, and form a colored complex. The 
absorbance of the complex was measured at 560 
nm. Since hydrogen peroxide content in many 
biological samples is much higher than that of 
other hydroperoxides, samples were pretreated 
with catalase to decompose the existing H2O2 
and eliminate the interference.   
The specific enzyme activities were expressed as 
U or mU/mg protein. LOOH concentration was 
expressed as nmol/mg protein. Protein 
concentration was determined by the method of 
Lowry et al. (9) and expressed as mg/ml. 
      Serum hormone levels were analysed in the 
hormone analysis laboratory. Estradiol (E) and 
progesterone (P) were determined by the RIA-
CT technique used for quantitative 
determination of E or P in human serum or 
plasma. Follicle-stimulating hormone (FSH) and 
luteinizing hormone (LH) were determined by 
the IRMA FSH and LH test, which determines 
the quantity of immune-radiometric human FSH 
and LH in serum. 
      Statistics. Statistical analysis was carried out 
by use of the Kruskal-Wallis test and the Dunn’s 
post hoc test, which considered the unequal and 
small sample sizes we used in this study. A linear 
regression model was used to evaluate 
associations between hormonal and antioxidant 
variables. Before plotting the data in the 
regression study, the normality test on the 
variables was performed and the values of 
estradiol and progesterone were log-transformed. 
Two-tailed p values are given throughout. All 
data were analyzed using GraphPad Prism 
software.  
Results 
Significant changes were observed for all 
hormone concentrations, FSH (H=5.99, p<0.05), 
LH (H=7.11, p<0.05), estradiol (H=8.10, 
p<0.05), and progesterone (H=6.42, p<0.05), 
(Table 1). We also found significant phase-
related changes of GPx (H=7.84, p<0.05) and 
468
GR activity (H=8.10, p<0.05). Both enzymes had 
similar activity pattern, which was higher in 
follicular phase compared to luteal phase and to 
postmenopause, (p<0.05). The phase-related 
activity of SOD and CAT as well as the LOOH 
level did not show any statistical difference 
(Table 2). 
The linear regression analysis of individual 
hormonal variables against antioxidant 
parameters in endometrium showed a 
significant negative correlation between 
FSH/LH concentrations and GPx activity (r= -
0.36; r= -0.47, p<0.05, respectively). 
 
Table 1. Hormone levels during follicular phase, luteal 
phase and in postmenopause (Data are expressed as 
mean ± SEM; * p<0.05). 
 
Table 2. LOOH concentrations and AO enzyme 
activities in follicular phase (F), luteal phase (L) and 
postmenopause (PM) in hyperplasia complex tissue. 
Data are shown as mean ± SEM. P values refer to the 
results of the Dunn test.  
 
 
 
 
Discussion 
Previous studies of AO enzymes in 
endometrium pointed to hormone-dependent 
expression and activity. Ota et al. (10) have 
shown the phase-dependent changes of  both 
forms of SODs (copper, zinc SOD and 
manganese SOD) in glandular and surface 
epithelia of healthy fertile women during the 
menstrual cycle. Specifically, their expression 
was lowest during the early and midproliferative 
phases, then gradually increased and was most 
pronounced in the early and mid-secretory 
phases. In women with endometriosis and 
adenomyosis, the expression of both SODs was 
constantly elevated. Some data also indicated 
that SOD activity was not different in 
postmenopausal women compared to fertile 
ones (11). Our recent findings in women with 
endometrial polyp showed the opposite pattern  
of LOOH concentration and SOD activity in 
polyp tissue than in women with hyperplasia 
complex. Both parameters were higher in the 
proliferative phase compared to the secretory 
phase or to the women in postmenopause (7). 
The results of this study showed that women 
with endometrial hyperplasia complex had 
similar SOD activity pattern like the healthy 
ones. Although it was not statistically significant, 
SOD activity slightly increased in luteal 
(secretory) phase.  
Regarding GPx, the studies shown that in 
normal human endometrium the amount of this 
enzyme was low in the early proliferative phase, 
gradually increased and reached a maximum in 
the late proliferative/early secretory phases, and 
decreased thereafter (12). In this study, however, 
the GPx activity in hyperplastic tissue had the 
opposite pattern than in healthy women. The 
same variation of activity was observed for GR. 
Ohwada et al. (13) also found that GPx activity is 
significantly higher in endometrial cancer tissue 
with atypical or mixed atypical adenomatous 
hyperplasia than in endometrial cancer without 
hyperplasia, pointing to the role of AO status 
and ROS in hyperplastic changes that may lead 
to endometrial cancer.  
In this study we recorded a negative 
correlation between GPx and gonadotropins in 
women with endometrial hyperplasia complex, 
which may indicate the important role of these 
hormones in hyperplastic changes. Our previous 
research also showed a modulatory effect of 
gonadotropins on AO enzymes in women with 
endometrial polyp (7). Endometrial hyperplasias 
are generally considered as precancerous lesions 
and are treated either conservatively or surgicaly, 
and also by radiation therapy (14). The 
regression of hyperplastic to normal 
endometrium is the main purpose of any 
conservative treatment which is based on the 
administration of agents, like progestagens (15) 
 
 
F 
(n=10) 
 
L 
(n=10) 
PM 
(n=11) 
 
FSH  
(U/L)* 
 
 
5.97±1.42 
 
 
18.06±4.87 
 
 
25.59±5.62 
 
 
LH  
(U/L)* 
 
 
0.60±0.30 
 
 
6.07±1.70 
 
 
12.02±4.33 
 
 
Estradiol 
(pg/ml)* 
 
 
69.47±29.21 
 
 
73.07±21.81 
 
 
21.27±3.75 
 
 
Progesterone 
(nmol/l)* 
 
 
1.10±0.10 
 
 
5.97±0.68 
 
 
4.83±0.84 
 
 F L PM 
SOD 
(U/mg 
protein) 
0.47 ± 0.05 0.54 ± 0.07 0.66 ± 0.07 
 
CAT 
 
70.18 ± 10.54 
 
77.34 ± 10.73 67.52 ± 14.80 
(U/mg 
protein) 
 
GPx 
(mU/mg 
protein) 
 
37.44 ± 3.27* 
 
21.48 ± 1.81 
 
19.84 ± 1.60 
 
GR 
(mU/mg 
protein) 
 
5.96 ± 0.47* 3.41 ± 0.65 3.313 ± 0.42 
LOOH 
(nm/mg 
protein) 
0.32 ± 0.04 0.34 ± 0.03 0.35 ± 0.02 
469
or gonadotropin-releasing hormone analogues 
(GnRHa) (16). Since the effects of these 
therapies are associated with AO and hormonal 
changes, our results may contribute to the 
prediction of potential therapeutic efficacy and 
to selection of the most effective treatment for 
hyperplasia complex. 
Acknowledgement 
This work was financially supported by the Ministry 
of Education, Science and Technological 
Development (Grants 41027, 41022, 173041). 
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Antioxidant Enzymes in Women with Hyperplasia Complex: Relation with Sex Hormones

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