143

OVARY DAMAGE

Biochemical and histopathological investigation of the protective effect of disulfiram in ischemia-induced ovary damage

Unal Isaoglu1, Mehmet Yilmaz1, Muhammet Calik2, Beyzagul Polat3, Ebubekir Bakan4, Ali Kurt5, Yavuz Albayrak6 & Halis Suleyman3

1Nenehatun Obstetrics and Gynecology Hospital, Erzurum, Turkey, 2Department of Medicinal Pathology, Faculty of Medicine, Atatürk University, Erzurum, Turkey, 3Department of Pharmacology, Faculty of Medicine, Atatürk University, Erzurum, Turkey, 4Department of Biochemistry, Faculty of Medicine, Atatürk University, Erzurum, Turkey, 5Department of Patholoy, Erzurum Regional Education and Research Hospital, Erzurum, Turkey, and 6Department of General Surgery, Erzurum Regional Education and Research Hospital, Erzurum, Turkey

Gynecological Endocrinology

2012

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10.3109/09513590.2011.589922

0951-3590

1473-0766

Gynecological Endocrinology, 2012; 28(2): 143–147Copyright © 2012 Informa UK, Ltd.ISSN 0951-3590 print/ISSN 1473-0766 onlineDOI: 10.3109/09513590.2011.589922

Correspondence: Prof. Halis Suleyman, Faculty of Medicine, Department of Pharmacology, Atatürk University, TR-25240, Erzurum, Turkey. Tel: +904422315202. Fax: +904422315202. E-mail: halis.suleyman@gmail.com

It was biochemically and histopathologically investigated whether disulfiram has protective effects on ischemia-induced ovary damage. For this purpose, levels of tGSH, superoxide dismutase (SOD), malondialdehyde (MDA), and 8-OH Gua/Gua were investigated in ischemic rat ovary tissue. Results show that used doses of disulfiram (10, 25, and 50 mg/kg) prevent MDA, a product of ischemia-induced lipid peroxidation, formation in female rat ovary tissue and prevent decrease of enzymatic and non-enzymatic (SOD, GSH) antioxidant parameters. Additionally, all doses of disulfiram significantly prevent DNA damage when compared to control group. Fewer histopathological findings were observed in tissues with higher antioxidant levels and lower oxidant and DNA damage levels.

Keywords: Disulfiram, ischemia, ovary

IntroductionOvarian ischemia is a serious pathological condition that appears in ovarian torsion by the obstruction of the vena and arteria ovarica. In ovarian torsion, the ovary is twisted or, in other words, it turns over around itself. Ovarian torsion constitutes 2.7% of all gynecological emergencies [1]. Ovarian torsion can be found in all age groups, including newborns. Delays in diagnosis and treatment can lead to ovarian deficiencies [2]. Ovarian torsion is generally seen in the right side; bilateral simultaneous torsion has not been reported in previous literature [3]. Vascular obstruction in torsioned ovary leads to massive congestion and hemorrhagic infarct in ovary parenchyma. Consequently, serious damage, such as gangrene and necrosis, occurs in the ovary [4].

Tissue damage starts with lipid radical formation in cell membranes, continues with lipid hydroperoxide conversion, and ends with genesis of toxic products (e.g., aldehyde, malon-dialdehyde (MDA) [5]. As a solution, organisms develop anti-oxidants as defense mechanisms against toxic oxygen radicals in tissues [6]. When these defense mechanisms fall short, free radicals damage not only lipids and proteins but also DNA molecules. In tissues, one indicator of DNA damage is 8-hydrox-iguanine (8-OH Gua). 8-OH Gua is an oxidation product of the DNA [7]. It has been experimentally reported that 8-OH Gua has a peak in the damaged tissues [8]. In ovarian torsion, because

clinical symptoms are nonspecific, diagnosis and treatment can be delayed [9]. The period between diagnosis and surgical initia-tive is a critical period that affects the prognosis of ovarian tissue in ischemic conditions. This can be explained by the fact that toxic oxygen radicals can peak in a short time period, such as 2 hours after the harmful agents enter [10]. For this reason, conser-vative treatment is necessary in the short time period between diagnosis and surgical invention in order to prevent the ischemic damage in ovarian tissue.

In the literature, there has been research on the cytoprotec-tive effect of disulfiram. In one study, the hepatoprotective effect of disulfiram after ischemia against cell death was shown [11]. Another study reported that disulfiram protects cells in tissue homogenate, which when obtained from dog spinal tissue homogenate against lipid peroxidation [12]. After reviewing the literature, there appears to be a lack of information on the effect of disulfiram on ischemic ovarian tissue. For this reason, the aim of our study is to investigate biochemically and histopathologically whether disulfiram has a protective effect in ischemia-induced ovarian tissue in rats.

Materials and methodsAnimals

A total of 30 female albino Wistar rats obtained from the Medical Experimental Research Centre, Ataturk University, weighing between 180 and 190 g were used in this study. The animals were fed and kept under normal conditions (22°C) in separate groups. Animal experiments were performed in accordance with the national guidelines for the use and care of laboratory animals and approved by the local animal care committee of Ataturk University.

Chemicals

For laboratory experimentation, sodium thiopental was obtained from IE-Ulagay Turkey, while disulfiram was purchased from Sigma Co., Germany.

Surgical procedure

All surgical procedures were performed on the rats under sterile conditions. Twenty-four rats were intraperitoneally anesthetized

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with 25 mg/kg thiopental. After injection, they were kept waiting for the appropriate time for surgical intervention. The proper time for surgery was determined by the self-immobilization of the animals in a supine position. A 2–2.5 peritoneal incision was applied vertically to reach the ovarian tissue. Then, the inferior part of the ovary was ligated and by this way ischemic condition was provided. The wound was closed after ligation, and animals were kept in a state of rest in the laboratory condition until they awoke. Six animals were used as intact control.

Application of drugs

Disulfiram was given to the rats two times: 1 hour before the thiopental injection, and 12 hours after the operation in 10, 25, and 50 mg/kg doses by oral gavage. Distilled water was given to the ischemic control group and intact control group at the same volume. Rats were kept under ischemic conditions for 24 hours. At the end of the medical treatment, animals were sacrificed by decapitation. Their right ovarian tissues were removed and sent to Histopathology and Biochemistry laboratories for investigation. All results from the medication groups were compared with both control and healthy groups.

Biochemical analysis of ovarian tissue

Total glutathione (tGSH) analysis

The amount of GSH in the total homogenate was measured according to the method of Sedlak and Lindsay with some modi-fications [13]. 5,5-dithiobis (2- nitrobenzoic acid) (DTNB) was used as an chromogen, and it formed a yellow-colored complex with SH groups. The absorbance was measured at 412 nm using a spectrophotometer.

Superoxide dismutase (SOD) analysis

Measurements were performed according to Sun et al. [14]. The absorbance of the purple-colored formazan was measured at 560 nm.

MDA analysis

The concentrations of ovarian lipid peroxidation were determined by estimating MDA using the thio barbituric acid test [15]. The absorbance of the supernatant was measured at 532 nm.

Isolation of DNA from ovarian tissue

Ovarian tissue was drawn and DNA isolated using Shigenaga et al.’s modified method [16]. The absorbance was measured at 260 and 280 nm. Purification of DNA was determined as A 260/280 ratio 1.8.

DNA hydrolysis with formic acid

Approximately, 50 mg of DNA was hydrolyzed with 0.5 ml of formic acid (60%, v/v) for 45 min at 150°C [17]. The tubes were allowed to cool. The contents were then transferred to Pierce micro-vials, covered with Kleenex tissues cut to size (secured in place using a rubber band), and cooled in liquid nitrogen. Formic acid was then removed by freeze-drying. Before analysis by HPLC, they were re-dissolved in the eluent (final volume 200 µl).

Measurement of 8-hydroxy-2 deoxyguanine (8-OH Gua) with HPLC

The amount of 8-OH Gua and guanine (Gua) was measured by using a HPLC system equipped with an electrochemical detector (HP Agilent 1100 module series, E.C.D. HP 1049 A), as described previously [17, 18]. The amount of 8-OH Gua and Gua was analyzed on a 250 × 4.6 mm Supelco LC-18-S reverse-phase column. The 8-OH Gua levels were expressed as the number of 8-OH Gua molecules/105 Gua molecules [19].

Histological examination

At the end of each experiment, the ovaries 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 4 μm and stained with hematoxylin and eosin. The histologic sections were examined for the presence of intersititial edema, vascular dilatation, hemorrahage and polymorphonuclear neutrophilic infiltrations, using a microscope Olympus BX-50 with a microscope and photographed. The slides were coded, and semiquantitative analysis of the ovary sections was performed without knowledge of treatment protocol. The changes seen were graded as follows: grade 0, normal; grade I, mild edema, mild vascular congestion, no hemorrhage and no leukocytic infiltra-tion; grade II, moderate edema, moderate vascular congestion, no hemorrhage and no leukocytic infiltration; grade III, severe edema, severe vascular congestion, minimal hemorrhage and minimal leukocytic infiltration; grade IV, severe edema, severe vascular congestion, hemorrhage and leukocytic infiltration.

ResultsThe effect of disulfiram on tGSH level, SOD activity and MDA level in ischemic ovarian tissue

As seen in Figure 1, the tGSH levels in ovarian tissues of the 10, 25, and 50 mg/kg given rats were 1.5, 3.3, and 5 nmol/g protein, respectively. While this level was 1.5 nmol/g protein in the isch-emic ovarian control group, it was 7.8 nmol/g protein in the healthy rat group.

The SOD activities in ovarian tissues of the 10, 25, and 50 mg/kg rats were 1.6, 2.8, and 4.0 μg, respectively. While this activity was 0.6 μg in the ischemic ovarian control group, it was 7.5 μg in the healthy rat group (Figure 1).

The MDA levels of the 10, 25, and 50 mg/kg given rat ovarian tissues were 11.1, 11.4, and 8.2 μmol/g protein, respectively. This level was 19.7 and 4.0 μmol/g in the ischemic ovarian control group and the healthy rat group, respectively (Figure 2).

The effect of disulfiram on oxidative DNA damage in ischemic ovarian tissue

In this measurement, the 8-OHGua/Gua level was measured as an indicator of DNA damage. As seen in Figure 2, the 8-OHGua/Gua levels in ovaian tissues of the 10, 25, and 50 mg/kg given rats were 13.5, 2.8, and 2.4 pmol/L, respectively. While this level was 4.8 pmol/L in the ischemic ovarian control group, it was 1.05 pmol/L in the healthy rat group.

Histopathological results

Ovarian tissues of healthy intact animals were regarded healthy (Figure 3). Serious edema, serious vascular congestion, slight hemorrhage and leukocyte infiltration were observed in animals that received 10 mg/kg disulfiram (Figure 4). In animals that received 25 mg/kg disulfiram, mediocre edema and vascular congestion, but no hemorrhage and leukocytes were observed (Figure 4). Ovaries of 50 mg/kg disulfiram administered animals looked like those of healthy animals, except slight edema and vascular congestion (Figure 4). Ovaries of ischemic control group animals were observed to have serious edema, serious vascular congestion, hemorrhage and leukocyte infiltration (Figure 3).

DiscussionIn this study, we biochemically investigated whether disulfiram had a protective effect in ischemia-induced ovarian damage in

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rats. For this reason, the effect of disulfiram was examined on tGSH, SOD, MDA, and 8 -OHGua/Gua as DNA damage indica-tors in ischemic rat ovarian tissue, and all tissues were histopatho-logically examined.

Our results have shown that 25 and 50 mg/kg doses of disul-firam significantly prevented a reduction in the tGSH level of ischemic ovary tissue when compared to the control group. Reduced glutathione (GSH) is an antioxidant that is found in

human and animal tissues [16]. GSH depresses superoxide radi-cals and protects protein thiol groups, which are considerably important for the cell integrity against oxidation [17]. GSH is used in the protection of intracellular protein, cysteine, dihy-drolipoate, coenzyme A, ascorbate alpha-tocopherol and in the reduction of ribonucleotide to form the deoxyribonucleoside precursor of DNA. In addition, GSH plays a role in the protec-tion of cells against oxidative damage and toxic compounds, and

Figure 3. Ovarian tissue of healthy intact rat group (left) and ischemic control group (right).

Figure 1. Effect of disulfiram on tGSH level and SOD activity in ischemic ovarian tissue. Bars represent mean values and error lines represent standard error.

Figure 2. Effect of disulfiram on MDA level and as a marker of DNA damage, 8-OH Gua/Gua levels level in ischemic ovarian tissue. Bars represent mean values and error lines represent standard error.

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in the metabolic functions of a variety of endogen compounds, such as prostaglandins and leukotrienes [20]. Glutathione reacts with free radicals directly and forms a substrate for peroxidase and glutathione-S-transferase. The antioxidant activity of GSH originates from its substrate function. For this reason, it is believed that in certain conditions (e.g., tissue ischemia/reperfusion) that cause production of excessive levels of toxic radicals, GSH decreases, and this decrease causes cell damage [21]. In this study, we also found that the GSH level was 5.2 times lower in the ischemic control group than in the healthy, intact rat group. Disulfiram of 25 and 50 mg/kg prevented GSH consumption in 35.9 and 57.7% of ischemic ovarian tissue, respectively. We observed that GSH was depleted in the destruc-tion of toxic radicals. Conservative invention performed before tissue reperfusion is important for damage reduction, because toxic radical production starts in ischemia and becomes severe in reperfusion [22].

In addition, in all doses (10, 25, 50 mg/kg), disulfiram significantly inhibited the decrease in SOD activity in isch-emic ovarian tissue when compared to the healthy group. We determined that disulfiram inhibited the consumption of SOD dose-dependently. In ischemic tissues, nearly 70% of oxygen is converted into superoxide radicals via NADPH-related oxidase [23]. SOD is an enzyme that catalyzes superoxide radicals into the non-toxic substances, hydrogen peroxide and molecular oxygen [24]. It was shown in a previous study that in ischemia-induced, damaged ovarian tissue, SOD activity was lower than the healthy rat group [25]. These previous findings are in accor-dance with our study.

In 10, 25, and 50 mg/kg disulfiram administered-ovarian isch-emic rats, the MDA level was lower than the ischemic control group. MDA is a lipid peroxidation product. Lipid peroxidation starts with a hydrogen atom extraction from polyunsaturated lipid chain in membrane via free radical in tissues. At the end of this reaction, the lipid acid gains a lipid radical func-tion. From this lipid radical, lipid peroxyl radicals are formed. Lipid peroxyl radicals oxidize membrane lipids and cause new radical production. MDA, which can be soluble in thiobarbi-turic acid, is produced via lipid acid peroxidation [26]. Since lipid radicals are hydrophobic, most of the reaction occurs in membrane-related molecules. Peroxyl radicals and MDA lead to serious damage by causing cross binding and polymerization of membrane components, and inactivating membrane recep-tors and membrane-binding enzymes [27]. In tissues with low

levels of lipid peroxidaton products (MDA) and other oxidants (MPO), GSH and other antioxidant parameters are high [28]. This information indicates that there is an important connec-tion between oxidant/antioxidant mechanisms and damage/repair of tissues.

As mentioned above, toxic radicals can damage not only lipids and proteins but also DNA molecules. In this study, we investigated whether disulfiram had a protective effect on DNA molecules of ischemic ovarian tissue. The reason for the study is to prove whether oxygen radicals can damage DNA in a similar manner to other tissues. For example, hydroxyl radicals cause DNA damage by extracting hydrogen from nucleic acid and reacting with double binds [30]. In our study, the protective effect of disulfiram was assessed by 8-hydroxyguanine (8-OHGua), a DNA damage product. 8-OHGua is accepted as one of the indi-cators of DNA oxidation [7]. In normal conditions, there is actu-ally a balance between oxidative damage and repair. This means that low levels of DNA damage can occur in healthy bodies [29]. In this study, 8-OHGua was determined to exist in low amounts, even in the healthy rat group. However, in ischemic groups, the 8-OHGua levels of rat ovarian tissues were 4.5 times higher than healthy ovarian tissues. In all doses, disulfiram significantly reduced levels of this DNA damage product when compared to the ischemic control group. This DNA oxidation product showed a decrease as the disulfiram dose increased. In other words, the highest dose of the disulfiram (50 mg/kg) was the most effective dose against DNA damage. Protective effect of disulfiram in ischemic ovarian tissue was supported by the results of histo-pathological examinations. Severity of histopathological findings (edema, vascular congestion, haemorrage, leukocyte infiltra-tion) increased as MDA levels and DNA damage increased. Less histopathological damage was observed in tissues with higher antioxidant levels. Previously, it was shown that in tissues with ischemia-induced damage, leukocyte infiltraion increased and the tissue underwent biochemical changes [30]. In conclusion, in all doses, disulfiram depressed lipid peroxidation under ischemic conditions in rat ovarian tissue, prevented the decrease in anti-oxidant parameters, and protected DNA from oxidation caused by toxic radicals. These findings were supported with histopatho-logical examinations.

Declaration of interest: None of the authors in this study had conflicts of interest, sources of financial support, corporate involvement, patent holdings, etc.

Figure 4. Ovarian tissue of disulfiram 10 mg/kg rat group (left), disulfiram 25 mg/kg rat group (middle) and disulfiram 50 mg/kg rat group (right).

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