Examination of the antioxidant effects of pre-HSG melatonin use on ovarian surface epithelium in rats : An experimental study

Material and methods. A total of 50 Wistar rats with regular estrous cycles were randomly divided into 5 groups. Group 1 was the control group. In other groups, X-ray was applied (group 2), 0.1 mL Lipiodol was applied to each uterine horn (group 3), 20 mg/kg intraperitoneal melatonin application was followed by 0.1 mL Lipiodol administration to each uterine horn after 15 min (group 4), and 20 mg/kg melatonin was administered to the ligamentum suspensorium ovarii, followed by 0.1 mL Lipiodol application to each uterine horn after 15 min (group 5). The rats in groups 2–5 were exposed to whole body radiation 3 times. After 3 h, the abdomens of all rats were reopened and left oophorectomy was performed.


Introduction
During the last decade, the number of women seeking infertility evaluation has increased significantly.Despite the development of other diagnostic procedures, such as magnetic resonance imaging, hysteroscopy and laparoscopy, hysterosalpingography (HSG) remains as the most valuable and commonly used gynecoradiological technique for the diagnosis of fallopian tube obstruction and uterine cavity abnormalities. 1 In HSG studies that are performed under fluoroscopic control, various radioscopic exposures are effectuated to visualize the fallopian tubes and the uterine cavity, following the gradual introduction of the contrast medium. 2 As HSG involves radiation exposure of the gonadal region in women of reproductive capacity and the possibility for repeated examinations, concerns over radiation doses and the associated radiation risks have become an issue. 3eople are constantly exposed to low levels of radiation because of the common use of certain electronic devices, air travel and medical diagnostic exams.Scientific and technological advancements have further increased the radiation burden in humans. 4onizing radiation consists of energetic particles and electromagnetic radiation, which can easily penetrate the living tissues or cells.The absorbed energy of ionizing radiation can break chemical bonds and cause the ionization of water and different biologically important macromolecules, such as nucleic acids, membrane lipids and proteins. 5Unstable and reactive free radicals, such as hydroxyl radical, superoxide anion and hydrogen peroxide, are produced more rapidly in ionized biological materials. 6These free radicals cause the peroxidation of membrane lipids, inactivation of enzymes, depolymerization of polysaccharides, and degradation of nucleic acids.Free radicals can cause severe irreversible damage to the cell membrane. 7ells are well equipped to defend themselves against free radicals, with antioxidant enzymes and molecules. 8These antioxidant systems consist of low-molecular-weight antioxidants, like glutathione and melatonin, and antioxidant enzymes, such as superoxide dismutase, catalase and glutathione peroxidase. 9he function of melatonin as an antioxidant and a free radical scavenger was first defined by Ianas et al. in 1991. 10elatonin, a derivative of amino acid tryptophan, is a neurohormone secreted from the pineal glands.However, other organs, such as the retina, ovaries, gastrointestinal tract, and lenses, are also capable of synthesizing melatonin. 11elatonin is considered a quite exceptional antioxidant, because it can easily cross the blood-brain barrier.Studies have shown that it neutralizes hydroxyl radicals, inhibits superoxide radicals through stimulating the mRNA levels for superoxide dismutase, increases glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase activities, and decreases the intracellular concentration of hydrogen peroxide. 12Lipiodol is iodized ethyl esters of poppy-seed oil fatty acids and has been in use as an iodinated contrast agent since 1926. 13It is a liquid, fiducial marker for image-guided radiation therapy (IGRT) used to mark the tumor beds. 14here is no study of whether the dysplastic changes in the ovarian surface epithelium of X-ray-exposed rats during HSG decrease or not with the use of Lipiodol and melatonin given both intraperitoneally and into the suspensorium ovarii.Therefore, the aim of this study was to histologically investigate the restorative effects of melatonin and Lipiodol administration during the HSG procedure on the dysplastic changes of the ovarian surface epithelium of rats.

Material and methods
Following the approval of the Ethics Committee of Firat University (Elazig, Turkey), all surgical procedures and care protocols used were planned and conducted in accordance with the "Guide for the Care and Use of Laboratory Animals".The experiments were performed on 50 female albino Wistar rats at the animal laboratory of Firat University.The rats were 5 months old, weighed approx.200-230 g and had regular ovulation cycles.They were kept at 21-23°C, with 55-60% relative humidity, under controlled photo-periods (12:12 h light:dark) and were fed with a standard rodent chow and tap water.Feeding of the rats stopped 18 h prior to the experiment, only water was allowed.The rats that were detected to be in the estrus phase via vaginal cytology were anesthetized with chloral hydrate (400 mg/kg, intraperitoneally -i.p.).Each rat was placed on the operating table in a supine position, and the abdomen was opened through a midline incision.The rats were randomized into 5 groups of 10 rats each: -group 1 (G1): the group in which the abdomen was opened and closed without any X-ray irradiation; -group 2 (G2): the group in which the abdomen was opened and closed, and X-ray was applied; -group 3 (G3): the group in which the abdomen was opened and 0.1 mL Lipiodol ® amp.(Guerbet Lab, Aulnaysous-bois, France) was applied to each uterine horn; -group 4 (G4): the group in which the abdomen was opened and 20 mg/kg melatonin (Melatonin ® , N-acetyl-5-metoksitriptamin; Sigma Chemicals, Deisenhofen, Germany) was administered i.p., followed by 0.1 mL Lipiodol ® application to each uterine horn after 15 min; -group 5 (G5): the group in which the abdomen was opened and 20 mg/kg melatonin (Melatonin ® , N-acetyl-5-metoksitriptamin; Sigma Chemicals) was administered to the ligamentum suspensorium ovarii, followed by 0.1 mL Lipiodol ® application to each uterine horn after 15 min.
After their abdomens were closed, the rats in G2, G3, G4, and G5 were exposed to whole body irradiation 3 times, leaving 2-minute intervals in between (total dose: 15-20 milirad, Villa-Genius HF-80 X-ray Generator; double table, double tube) (Del Medical, Milan, Italy).During the experiment, the blood pressure, heart rate and body temperature of the rats were monitored.After 3 h, the abdomens of all rats were reopened and left oophorectomy was performed.Samples from the left ovaries were embedded into paraffin blocks after fixing in 10% formaldehyde for histologic examination, and then sliced into 4-micrometer thick sections.
After the preparations were stained with hematoxylineosin, changes in the ovarian surface epithelium were examined according to the endometrium ablation scale. 15ccording to the scale, 1. stratification; 2. tufting; 3. chromatin irregularity; 4. nuclear contour irregularity; 5. increase in nucleus size; 6. increase in nucleus/cytoplasm ratio; 7. pleomorphism; 8. presence of nucleoli; 9. mitosis; and 10. hyperchromasia changes in the ovarian surface epithelium were examined.For each parameter, a scale (none = 0 points, present = 1 point, considerably present = 2 points) was applied, and the total score was obtained based on the values of all the parameters in a preparation.
The Kruskal-Wallis variance test from the SPSS Package Software (IBM, Armonk, USA) was used in the statistical analysis of data.The Mann-Whitney U test with Bonferroni correction was applied for parameters with p < 0.05.A probability value of p < 0.005 was considered statistically significant.

Results
The presence of nucleoli and mitosis values were found similar in all groups (p > 0.05, Kruskal-Wallis variance test).
All parameters except nucleoli and mitosis values increased significantly in the X-ray exposed group compared to other groups (p < 0.005, Mann-Whitney U test).
The detrimental effects of X-ray application diminished with Lipiodol use and were further reduced by the use of melatonin in combination with Lipiodol.
The presence of hyperchromasia and the total score were found to be the highest in group 2, followed by group 3, compared to other groups (p < 0.005, Mann-Whitney U test).All examined parameters are listed in Table 1.

Discussion
The ovaries are a part of the female reproductive system that is very sensitive to radiation.Ovarian damage is a major complication observed in patients who receive longterm radiotherapy.High doses of radiotherapy damage the ovarian function of all ages, causing a decline in fertility. 16herefore, researchers' interest in rat estrus cycle studies has increased in order to prevent ovarian damage caused by radiation, to discuss the effects of radiotherapy and to evaluate normal ovarian function.
The total number of follicles is regarded as one of the most important parameters reflecting ovarian function. 17ee et al. investigated the damage in primordial and primary follicles by collecting the ovaries of female prepubertal mice exposed to 8.3 Gy gamma radiation after 3, 6 and 12 h after irradiation. 18They reported that the most damage occurred after 3 h of irradiation, and the damage decreased as exposure time progressed.For this reason, in order to determine the dysplastic changes in the rat ovarian surface epithelium, the ovarian tissues were collected after 3 h of exposure and histologically evaluated for each group.Our study demonstrated that the radiation received during HSG treatment significantly increased stratification, tufting, chromatin irregularity, nuclear contour irregularity, nucleus size, nucleus/cytoplasm rate, pleomorphism, and hyperchromasia in the rat ovarian surface epithelium.Similarly, Pala et al. reported that markedly increased epithelial degeneration and obstruction was observed in the endometrial glands of rats that were exposed to X-ray irradiation with Lipiodol, as compared to other groups. 19Said et al. reported degeneration of the mucosal lining epithelial cell layers of the uteri in rats that were exposed to gamma radiation. 20Radiation triggers carcinogenic changes in the ovarian surface epithelium.Our results were similar to the histological changes observed in radiation-exposed normal cells. 21,22ur study confirmed that the detrimental effects of X-ray application in the rat ovarian surface epithelium were significantly diminished with Lipiodol application.This may be due to the fact that linoleic acid, an antioxidant constituent of Lipiodol, has a cytotoxic effect on macrophages, reducing the production of reactive oxygen species (ROS) in the ovaries and having antiproliferative activity.Sapmaz and Akpolat reported that Lipiodol use significantly diminished the dysplastic changes in luminal epithelial cells of rats that were exposed to X-ray irradiation. 23In previous studies, Lipiodol uptake by the cancer cells and by the endothelial cells was demonstrated on light and electron microscopy as early as 3 h after I-lipiodol application.Lipiodol is rapidly taken up and retained in the cells in the form of cytoplasmic bound vesicles. 24It also has a role as a vehicle to carry other agents inside the cells. 25Sapmaz et al. examined the effects of Lipiodol and Lipiodol combined with tetracycline against X-ray exposure during HSG treatment, and reported that Lipiodol use diminished the carcinogenic effects of radiation on ovarian surface epithelium, and were further reduced by the use of tetracycline in combination with Lipiodol. 15][28] During antioxidant defense, melatonin not only neutralizes hydroxyl radicals, but also scavenges peroxyl radicals and singlet oxygen throughout peroxidation. 29This implies that melatonin, which is lipophilic and hydrophilic, has effects not only at the cellular level, but also in the subcellular compartments. 30Melatonin stimulates the activity of the detoxifying enzyme glutathione peroxidase and strengthens the antioxidant defense of the cells against oxidative stress. 31hat is how melatonin stimulates antioxidant defense or exhibits strengthening actions against ionizing radiation which causes genetic damage by increasing the production of free radicals. 32In our study, changes in ovarian surface epithelium in rats treated with Lipiodol and melatonin were found to be similar to the control group.Sapmaz and Akpolat reported that the use of melatonin in combination with Lipiodol has a decreasing effect on congestion, endometrial ablation, fibrosis, and dysplastic changes to the control levels in X-ray-treated rats. 23It is thought that this decreasing effect is due to the stimulation of the antioxidant defense system of the cells against the damage caused by ionizing radiation by elevated intracellular concentrations of melatonin with Lipiodol's action.Radiation causes arteritis in large vessels such as coronary, pulmonary, thoracic aorta, brachial, renal, and ilio-femoral vessels, and causes hypoxia and ischemia in the perfused organs. 33,34In our study, the rats in the X-ray group may have developed a more severe radiation-associated ilio-femoral or uterine arteritis than the rats in the groups G3, G4 and G5.This may have led to a severe hypoxic effect and an increase in ROS production in G2, leading to the onset of carcinogenic changes in the ovarian surface epithelium.Even though other groups developed radiation-associated ilio-femoral or uterine arteritis of similar severity, it is thought that the combined use of Lipiodol and melatonin, which have antioxidant properties, was effective in reducing the detrimental effects of radiation.The study conducted by Badr et al. on rats confirmed that melatonin treatment 1 h before the X-ray irradiation prevented chromosomal damage, but no significant effect was observed when melatonin was given 1/2 h after the exposure. 30Undeger et al. demonstrated that intraperitoneal pretreatment of rats with 100 mg/kg melatonin provided a significant decrease in the DNA strand breakage and lipid peroxidation in the brain tissue. 35Canyılmaz et al. reported that 100 mg/kg intraperitoneal melatonin administration 1/2 h prior to a single dose of 6-Gy-gamma irradiation decreased the tubular atrophy and lipid peroxidation in the rat kidney tissues. 36Sato et al. conducted a study in order to reduce the risk for postoperative acute liver failure, in which prostaglandin was administered either from the hepatic artery or superior mesenteric artery to patients who were undergoing major hepatic resections, and found that prostaglandin applied through the hepatic artery was infused more rapidly. 37In our study, melatonin was administered in 2 different ways prior to irradiation, intraperitoneally (G4) and into the suspensorium ovarii (G5).Although both groups were at the control level, it was determined that the values of G5 were better than those of G4.This suggests that antioxidants may increase their protective effects if administered closer to the target organs.Pala et al. reported that there was no significant difference in epithelial degeneration and congestion in rats administered with Lipiodol combined with vitamin C and Lipiodol combined with vitamin E i.p. prior to irradiation, indicating that both groups were at the control level. 19Sapmaz et al. reported that melatonin was more effective than oxytetracycline in their comparative study of the antioxidant effects of melatonin and oxytetracycline in autologous intraperitoneal ovary transplantation. 38Histoenzymological and morphological studies suggested that melatonin was shown to act synergistically with chemotherapeutic agents or radiotherapy in order to increase the antitumor effects by promoting disease stabilization. 39Therefore, it is believed that melatonin and Lipiodol administration to patients prior or during the HSG procedure can reduce the harmful effects of radiation.Based on the results of our work, it is thought that melatonin and Lipiodol administration may prevent the carcinogenic changes exerted by radiation on the ovarian surface epithelium.

Table 1 .
Examined parameters of all groups with respect to the changes in the ovarian surface epithelium Values shown are means ± standard deviation (SD).The letters a-c indicate statistical differences among the groups (p < 0.005, Mann-Whitney U test); * groups with parameters significantly different from control (G1).