Genistein attenuates postischemic ovarian injury in a rat adnexal torsion-detorsion model

Genistein attenuates postischemic ovarian injury in a rat adnexal torsion-detorsion model Gurkan Yazici, M.D.,a Ozlem Erdem, M.D.,b Burak Cimen, M.D.,c Murat Arslan, M.D.,a Bahar Tasdelen, Ph.D.,d and Ismail Cinel, M.D.e a

Department of Obstetrics and Gynecology, School of Medicine, Mersin University, Mersin, Turkey; b Department of Pathology, School of Medicine, Gazi University, Ankara, Turkey; c Department of Biochemistry, d Department of Biostatistics, and e Department of Anesthesiology and Reanimation, School of Medicine, Mersin University, Mersin, Turkey

Objective: To investigate the effects of genistein on reperfusion injury in a rat ovarian torsion-detorsion model. Design: Controlled experimental study. Setting: University animal research laboratory. Subject(s): Thirty-two Wistar-Albino rats. Intervention(s): The rats were divided into four groups. Sham operation was performed in group I. In group II, 5 mg/kg genistein was given intraperitoneally (IP) during laparotomy, and right ovaries were removed 4 hours later. In group III, right ovaries were subjected to 4 hours of adnexal ischemia by use of vascular clips, and after ischemic insult, 4 hours of reperfusion was maintained by removing the clips. In group IV, after the ischemic period, 5 mg/kg genistein was given IP, and 4 hours of reperfusion was maintained. Right ovaries were surgically removed at the end of the procedure in each group. Main Outcome Measure(s): Ovarian histopathologic findings were scored and compared among study groups. Serum and ovarian tissue malondialdehyde (MDA), glutathione peroxidase, and superoxide dismutase, levels were measured. Result(s): Ovarian tissue damage scores were significantly different among groups and were seen to correlate with ovarian tissue MDA levels. Genistein significantly decreased the tissue damage scores, ovarian tissue MDA levels, and serum MDA levels. Conclusion(s): Genistein attenuates ischemia-reperfusion injury in rat adnexal torsion-detorsion model. (Fertil Steril威 2007;87:391– 6. ©2007 by American Society for Reproductive Medicine.) Key Words: Genistein, ischemia-reperfusion, adnexal torsion, malondialdehyde

Adnexal torsion is a rare but serious cause of gynecologic surgical emergency with a prevalence of 2.7% (1). Although immediate surgical treatment is required to preserve fertility and salvage the adnexa, nonspecific symptoms and clinical findings delay the diagnosis. Traditionally, where an ischemic twisted adnexa with a “black-bluish” appearance was found, salpingo-oophorectomy without untwisting the adnexa was preferred to avoid potential thrombotic emboli from the ovarian vein (1–3). However, recent reports have advocated a conservative approach of releasing the pedicle and evaluating the tissue reperfusion (3, 4). After detorsion and maintaining the circulation of the ovary, a pathologic process called reperfusion injury is encountered. Reperfusion of ischemic tissue, although necessary for reparative mechanisms, has been shown to worsen acute ischemic injury via the release of reactive oxygen species (ROS). Both ischemia and reperfusion (reintroduction of oxygen to hypoxic tissue) are important in human pathophysiology because they occur in a wide variety of important clinical conditions. Prominent examples of tissue hypoxia that preReceived January 3, 2006; revised and accepted June 30, 2006. Reprint requests: Gurkan Yazici, M.D., B.H. Pasa Bulvarı, Liparis plaza, Orkide blok, Kat:5, D:12, Mezitli, Mersin, Turkey (FAX: ⫹90 324 337 43 05; E-mail: [email protected]).

0015-0282/07/$32.00 doi:10.1016/j.fertnstert.2006.06.056

dispose to injury during reoxygenation include circulatory shock, myocardial ischemia, stroke, and transplantation of organs (5). Ischemia and consecutive reperfusion causes oxidative stress, which is characterized by an imbalance between ROS and the antioxidative defense system. ROS have been implicated in the pathogenesis of the structural and functional alterations of the tissues that are associated with a variety of pathologic processes (5). Antioxidant therapies used to overcome ischemia-reperfusion–related phenomena include the use of N-acetylcysteine, vitamin E, vitamin C, superoxide dismutase, catalase, and allopurinol. It has been shown that prevention of reperfusion injury increases the success of the treatment in such states (6, 7). Genistein (4=,5,7-trihydroxyisoflavone) is a common precursor in the biosynthesis of antimicrobial phytoalexins and an important phytoestrogen with a wide variety of pharmacologic effects, including tyrosine kinase inhibition (8, 9). Most of the studies have focused on the pharmacologic activities of genistein as a tyrosine kinase inhibitor, its chemoprotectant activities against cancers and cardiovascular disease, and its antioxidant and phytoestrogen activity (9). The aim of this study is, therefore, to determine the effects of genistein on the ovarian morphology, lipid peroxidation, and antioxidant enzymes in a rat adnexal torsiondetorsion model.

Fertility and Sterility姞 Vol. 87, No. 2, February 2007 Copyright ©2007 American Society for Reproductive Medicine, Published by Elsevier Inc.

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MATERIALS AND METHODS Thirty-two female Wistar rats at 90 days of age weighing 250 –300 g were used in the study. The mean age and body weight of all animals were identical. Surgical procedures described below were performed in accordance with the National Institutes of Health approved guidelines. The study protocol was reviewed and approved by our institutional ethics committee. Rats were randomly divided into four groups: group I control group (n ⫽ 8) (sham operated) laparotomy alone; group II (n ⫽ 8) laparotomy and genistein; group III (n ⫽ 8) ischemia-reperfusion; and group IV (n ⫽ 8) ischemia-reperfusion and genistein. Surgical Procedure Each rat was weighed and anesthetized with ketamine hydrochloride 60 mg/kg IM (Eczacibasi, Istanbul, Turkey), which was repeated as necessary to maintain anesthesia. Rats were placed in dorsal recumbent position, and the skin area of the incision was cleaned and dressed. A 2-cm midline incision was used for laparotomy, and the uterine horns and adnexa were located. Sham operation was performed in group I. In group II, 5 mg/kg genistein (dissolved in dimethyl sulfoxide) was given intraperitoneally (IP) during laparotomy, and ovaries were removed 4 hours later. In groups III and IV, 4 hours of adnexal ischemia was produced in the right ovaries by using vascular clips, which has been described previously in rats (6, 10). In group III; after duration of ischemia, 4 hours of reperfusion was applied by removing the clips, and ovaries were removed for examination. In group IV, after the ischemic period, 5 mg/kg genistein was administered IP, and reperfusion was maintained by removing the vascular clips. After 4 hours of reperfusion, right ovaries were surgically removed. A part of the ovarian tissue was preserved in formalin for histologic examination. After washing with 0.9% NaCL, the remaining part was stored at ⫺30°C until the biochemical analysis to determine the tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity. Blood samples were obtained at the end of the procedure in each group of animals. Histologic Examination The ovarian tissues were removed and fixed in 10% neutral buffered formalin solution and then embedded in paraffin as usual. Serial sections were cut using the microtome at a thickness of 5 ␮m and stained with hematoxylin and eosin. The histologic sections were examined for the presence of ischemiareperfusion injury with a scoring system that has been reported previously (6). Congestion, hemorrhage, interstitial edema, and loss of cohesion were scored from 0 to 3 according to their severity, where 0 represents no pathologic finding, and 1, 2, and 3 represent pathologic findings of ⬍33%, 33%– 66%, and ⬎66% of the ovary, respectively. The scores for each parameter were summed, and the total tissue damage scores were calculated. An Olympus BX-50 light microscope (Olympus Inc., 392

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Tokyo, Japan) was used, and the pieces were photographed. Analysis of the ovary sections was performed in a blinded fashion by the same pathologist. Biochemical Analysis Tissues (which had been preserved at ⫺30°C) were homogenized in 1 mL 0.9% NaCl using a tissue homogenizer. The homogenates were centrifuged at 1,500 ⫻ g at 4°C for 10 minutes to remove insoluble cellular debris. Upper supernatant fluid was removed, and assays were performed in this fraction. All the procedures were performed at ⫹4°C throughout the experiments. Protein was measured by the Lowry procedure (11) with serum bovine albumin as the standard. SOD, GSH-Px, and catalase (CAT) enzymes activities were measured as described in Refs. 12, 13, and 14, respectively. One unit of SOD activity is defined as the enzyme protein amount causing 50% inhibition in nitrobluetetrazolium reduction rate, and results are expressed as U/mL for sera samples and U/mg protein for ovarian tissues. The GSH-Px activity method is based on the measurement of the absorbance decrease at 340 nm due to the consumption of reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH). GSH-Px activities are expressed as IU/mL for sera samples and IU/mg protein for tissues. CAT enzyme activities are based on the measurement of the absorbance decrease due to H2O2 consumption at 240 nm, and results are expressed as IU/mL. MDA activity method is based on the spectrophotometric absorbance measurement of the pinkcolored product of thiobarbituric acid–malondialdehyde complex formation at 532 nm, and results are expressed as nmol/mL for sera samples and nmol/mg protein for ovarian tissues (15). Statistical Analysis The Statistical Package for Social Sciences (SPSS Inc., Chicago, IL), version 10.0, was used for statistical analysis. Individual group biochemical parameters were assessed with one-sample Kolmogorov-Smirnov Z test and found normal (P⬎.05). Analysis of variance was performed on the biochemical data to examine differences among groups. If a significant group effect was found, a Duncan’s multiplerange test was used to identify the location of differences between groups. Statistical significance was defined as P⬍.05. Tissue damage scores were compared by nonparametric analysis, and statistical significance was determined by Kruskal-Wallis followed by Bonferroni corrected Mann-Whitney U test (␣1 ⫽ 0.0083). Pearson correlation analysis was used between biochemical parameters and total tissue damage scores. The results are given in the text as means ⫾ SD. RESULTS Ovarian sections were assessed for tissue damage by histologic examination. Figure 1A shows a representative photomicro-

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FIGURE 1 Pathologic examination of the ovaries from group I (A) and group II (B) had a normal appearance. Ovarian sections after ischemia and reperfusion in group III (C) show significant pathologic changes (infiltration by neutrophils, diffuse congestion, hemorrhage, edema, and loss of cohesion) that reduced by intraperitoneal administration of genistein in group IV (D). Hematoxylin and eosin; original magnification, ⫻100.

Yazici. Effect of genistein on adnexal torsion-detorsion. Fertil Steril 2007.

graph of an ovarian section from a sham-operated animal. There was no evidence of epithelial disruption. Histopathologic assessment of the ovaries from group I and group II revealed normal appearance (Fig. 1A and B). Figure 1C obtained from a representative rat subjected to ovarian ischemiareperfusion demonstrates the presence of moderate morphologic damage indicated by the appearance of massive hemorrhage, acute infiltration by polymorphonuclear neutrophils (PMNs), diffuse congestion, intersititial edema, and loss of cohesion. Genistein administration reduced the pathologic changes induced by ischemia-reperfusion (Fig. 1D); in particular, PMN infiltration, hemorrhage, and loss of cohesion were much lower but had not been totally prevented. Total tissue damage scores as described in Materials and Methods are summarized in Figure 2. Total tissue damage Fertility and Sterility姞

scores were significantly different among groups (P⫽.000). Rats in the ischemia-reperfusion group had significantly higher histologic score (9.3 ⫾ 1.6) compared with group I (2.0 ⫾ 2.2; P⫽.000), group II (1.7 ⫾ 1.8; P⫽.000), and genistein-treated group IV (4.9 ⫾ 3.2; P⫽.007). Tissue damage scores were seen to correlate with ovarian tissue MDA levels (r ⫽ 0.487, P⫽.003). Tissue and serum MDA, GSH-Px, SOD activity levels and serum CAT activities are given in Table 1. Ovarian tissue and serum MDA levels were significantly different among groups (P⫽.000). Ovarian tissue and serum MDA levels in group III were significantly elevated; more than those in group I, group II, and group IV (P⬍.05). The serum GSHPx, SOD, and CAT levels and tissue GSH-Px and SOD levels were comparable among groups (P⬎.05; Table 1). 393

FIGURE 2 Total tissue damage scores were significantly different among groups (P⫽.000). Tissue damage scores (means ⫾ SD): group I, 2.0 ⫾ 2.2; group II, 1.7 ⫾ 1.8; group III, 9.3 ⫾ 1.6; group IV, 4.9 ⫾ 3.2. Rats in group III had significantly higher histologic scores compared with group I, group II, and group IV (P⫽.000).

the maintenance of ovarian morphology. Although our results do not suggest a direct relationship between the levels of antioxidant enzymes and lipid peroxidation in ovarian tissue, the protection of ovarian morphology by genistein pretreatment might be due to the inhibition of lipid peroxidation in the ischemia-reperfusion group, clearly demonstrating the involvement of oxidant-antioxidant balance. Although adnexal torsion may occur at any age, the majority of patients are in the reproductive age group (1). Detorsion and adnexal conservation would have advantages especially in the reproductive age group instead of oophorectomy (3). Delay in diagnosis of adnexal torsion resulting in irreversible tissue necrosis is a factor contributing to ovarian loss. The length of time of a failure in the blood supply to the adnexa before the occurrence of irreversible damage is unknown. Clinical inspection is unreliable in differentiating reversible tissue ischemia from irreversible tissue necrosis (3, 10). The ischemic tolerance of the ovary has not been determined clearly. There are conflicting reports in animal studies. One group found no irreversible histologic change after 4 –24 hours of ischemia and 7 days of reperfusion (10). Change in tissue concentrations of radical scavenger activity was not significant in 4- to 24-hour groups. However, these authors did not measure MDA levels within the ischemic tissue (10). Other studies have clearly demonstrated histologic changes after 1, 3, and 4 hours of ischemic duration and additional reperfusion injury after detorsion with significant elevation of tissue MDA levels (6, 16, 17).

Yazici. Effect of genistein on adnexal torsion-detorsion. Fertil Steril 2007.

DISCUSSION In the current study, we demonstrated that genistein treatment attenuates ischemia-reperfusion–induced lipid peroxidation and prevents postischemic ovarian injury. Genistein treatment reduces lipid peroxidation, which contributes to

Increased concentrations of MDA reflect the level of lipid peroxidation in tissues, and it is considered a marker of tissue injury (16). In the current study, the levels of tissue MDA, an index of lipid peroxidation, were significantly decreased by intraperitoneal administration of genistein. It was already demonstrated that genistein was able to inhibit low-density lipoprotein (LDL) oxidation, endothelial cell proliferation (9), and inhibits protein tyrosine kinase (18). Also, genistein limits the inflammatory response and neutrophil infiltration, which might be regarded as another source

TABLE 1 Ovarian tissue and serum malondialdehyde, glutathione peroxidase, and superoxide dismutase activity levels and serum catalase levels. Group I

Group II

Group III

Group IV

MDA (Ovarian tissue) (nmol/mg ⫾SD) 172.9 ⫾ 96.5 104.2 ⫾ 98.7 307.5 ⫾ 76.4 134.1 ⫾ 64.8 MDA (Serum) (nmol/mL ⫾SD) 4.8 ⫾ 1.5 4.2 ⫾ 1.1 7.2 ⫾ 1.9 5.3 ⫾ 1.8 GSH-Px (Ovarian tissue) (IU/mg ⫾SD) 5.7 ⫾ 1.3 5.5 ⫾ 1.6 4.4 ⫾ 0.9 5.2 ⫾ 1.9 GSH-Px (Serum) (IU/mL ⫾SD) 0.49 ⫾ 0.15 0.45 ⫾ 0.16 0.57 ⫾ 0.14 0.51 ⫾ 0.15 SOD (Ovarian tissue) (U/mg ⫾SD) 80.1 ⫾ 37.6 90.9 ⫾ 47.1 84.5 ⫾ 19.8 92.0 ⫾ 43.8 SOD (Serum) (U/mL ⫾SD) 7.5 ⫾ 2.7 8.2 ⫾ 2.7 9.9 ⫾ 0.5 9.2 ⫾ 0.6 CAT (Serum) (IU/mL ⫾SD) 264.4 ⫾ 178.3 270.8 ⫾ 243.4 193.1 ⫾ 79.7 240.6 ⫾ 145.6

P value .001 .007 .37 .43 .93 .10 .80

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of free radicals in the ischemic tissue, as activation of neutrophils results in the production and release of toxic oxygen metabolites (19). The major tissue damage that occurs during ischemiareperfusion injury is secondary to calcium influx into the cell (20). The Ca2⫹ overload is induced mainly by the action of sarcolemmal Na⫹/H⫹ and Na⫹/Ca2⫹ exchangers, which then activate several intracellular processes (21). Transient, but large, cellular and mitochondrial Ca2⫹ fluxes occur during hypoxia-reoxygenation. Reoxygenation causes significant elevation in intramitochondrial Ca2⫹. Release of Ca2⫹ from storage sites stimulates Ca2⫹-dependent proteases, nucleases, and phospholipases. Increased intracellular Ca2⫹ also appears to be related to oxidation of adenine nucleotides (5). Studies suggest that transient current and L-type Ca2⫹ current are regulated by tyrosine phosphorylation (22, 23). For example, in rabbit colonic smooth muscle cells, cytoplasmic tyrosine kinase can regulate L-type Ca2⫹ current by direct phosphorylation of the ␣1-subunit of the Ca2⫹ channel (23). Furthermore, genistein was able to inhibit L-type Ca2⫹ channel (24, 25). Bcl-2 overexpression attenuates lipid peroxidation induced by various kinds of agents and protects cells or facilitates their recovery from hydrogen peroxide–induced oxidative DNA damage (26). Overexpression of Bcl-2, which has antioxidant properties, in mitochondrial membranes inhibits Ca2⫹ efflux due to oxidants (5). These findings suggest the distinct role of Bcl-2 in regulation of cellular redox state against oxidative stress (26). In cortical neuronal cell lines, genistein was able to prevent the downregulation of the anti-apoptotic protein Bcl-2 that was caused by oxidative agent. These results indicate that genistein has a protective effect in cortical cells, which was mediated by its regulation of Bcl-2 (27). Fas is a ubiquitous receptor belonging to the tumor necrosis factor–nerve growth factor superfamily and is activated by Fas ligand (FasL), which can cause apoptosis in Fas-bearing cells (28). In several important diseases, Fas activation results in apoptotic, as well as in non-apoptotic effects. Nevertheless, recent studies have shown that Fas activation is involved not only in pathologies inflicted by immune effectors but also in lymphocyte-independent diseases, such as ischemia-reperfusion injuries (28). Expression of both Fas and FasL was demonstrated in granulosa cells and oocytes of rat ovary and in female gonads (29, 30). Tyrosine phosphorylation has been shown to participate in Fas receptor signaling in various cellular systems including germ cells (29, 31). Indeed, in support of this hypothesis, Shilkrut et al. demonstrated that tyrosine kinase blockade by genistein prevents Fas-mediated nonapoptotic deleterious effects in rat hypoxic ventricular myocytes (22). Fas activation leads to intracellular calcium concentration rise, which was blocked by tyrosine kinase inhibitor, genistein. They concluded that tyrosine phosphorylation is a critical step in Fas-induced damage (22). Fertility and Sterility姞

Genistein is structurally similar to 17B-estradiol, binds to estrogen receptors, and exhibits estrogenic properties. Estrogen has been postulated as a “survival factor” for endothelial cells (32). The beneficial effect of estrogen in acute ischemiareperfusion injury has been demonstrated in liver (33), cardiac myocytes (34), intestines (35), and neuronal tissue of Wistar rats (36). Interestingly, the beneficial effects of estrogen therapy were not completely reversed by the selective estrogen receptor antagonist ICI-182,780 (33). Both estrogen and genistein can rapidly induce the release of endothelial cell nitric oxide synthase (eNOS)-derived nitric oxide (NO) via a nongenomic manner (32, 37). NO is known to inhibit ROS-mediated reactions, and it has been suggested that the protective effects in a variety of conditions are due to the ability of NO to detoxify ROS such as O2⫺, OH·, and/or ferryl hemoprotein (38). This might be one of the main causes why ovarian tissue MDA content was significantly lower in group IV than in group III. Selective regulation of mitogen-activated protein kinase (MAPK) activities and inhibitory effects on tumor necrosis factor-␣ (TNF-␣) production might also take place in this protective role of estrogens and genistein against ischemiareperfusion injury (19, 33, 34, 36, 39). Deodato et al. demonstrated that genistein reduced the serum levels of TNF-␣ in rats subjected to myocardial ischemia-reperfusion injury (19). Recently, the protective effect of another phytoestrogen, resveratrol, was been clearly indicated in an ovarian ischemia-reperfusion model (40). Our findings suggest that genistein attenuates ischemiareperfusion injury in the rat ovarian torsion model. This result is in agreement with previous experimental models showing that genistein reduced reperfusion injury in various tissues (18, 19, 25, 27, 41). Precise mechanisms of adnexal injury after ischemia and reperfusion are not fully understood. Also, it seems that antioxidative enzymes are not the unique preventive mechanisms of cellular injury after ischemiareperfusion of the specific tissue. Possible mechanisms underlying this protective effect of genistein were discussed above, and genistein might take a place in the management of adnexal torsion in the future. We believe that further studies regarding the exact mechanisms of ischemiareperfusion injuries will provide new insights in therapeutic models of oxidative stress including adnexal torsion. REFERENCES 1. Hibbard L. Adnexal torsion. Am J Obstet Gynecol 1985;152:456 – 61. 2. Pryor R, Wiczyk HP, O’Shea DL. Adnexial infarction after conservative surgical management of torsion of a hyperstimulated ovary. Fertil Steril 1995;63:1344 – 46. 3. Bayer AI, Wiskind AK. Adnexal torsion: can the adnexa be saved? Am J Obstet Gynecol 1994;171:1506 –10. 4. Oelsner G, Bider D, Goldenberg M, Admon D, Mashiach S. Long-term follow-up of the twisted ischemic adnexa managed by detorsion. Fertil Steril 1993;60:976 –79. 5. Li C, Jackson RM. Reactive species mechanisms of cellular hypoxiareoxygenation injury. Am J Physiol (Cell Physiol) 2002;282:C227– 41.

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