Abstract
Methotrexate (MTX) has toxic effects on the uterus and ovaries via oxidative stress. Coenzyme Q10 (CoQ10) is an important component in electron transport in the mitochondria and an antioxidant in cellular metabolism through the inhibition of lipid peroxidation. The aim of this study was to investigate the preventive effects of CoQ10 on MTX-induced utero-ovarian damage and oxidative stress in rats.
In this experimental study, 30 albino Wistar female rats were divided randomly into three groups. Once a day for a month, 10 mg/kg of CoQ10 was orally administered to the rats in the MTX+CoQ10 group, while the same volume of olive oil was administered orally to the other two groups. One hour thereafter, 20 mg/kg of MTX was injected intraperitoneally into the rats in the MTX and MTX+CoQ10 groups; the remaining group was the control. At the end of the month, biochemical and histopathologic examinations were performed on the extracted uteri and ovaries. In the uterine ovarian tissues of the animals in the MTX group, there was an increase in oxidative stress mediators and a decrease in antioxidant and anti-inflammatory mediators, but these trends were reversed in the MTX+CoQ10 group, demonstrating the antioxidant effects of CoQ10. MTX leads to oxidative stress-related ovarian and uterine injury, and CoQ10 may be useful for protecting ovarian and uterine tissue from such injury.
Introduction
Methotrexate (MTX) is a chemotherapeutic folic acid antagonist that is used in the treatment of diseases1–3—particularly inflammatory and autoimmune diseases2–4—but, at high doses, it has serious toxic effects on organs and tissues. Increased levels of reactive oxygen species, oxidative stress, and inflammatory processes have emerged as key players in the pathogenesis of MTX-induced damage to organs and tissues,5–7 including ovarian functional and structural disorders and, at high doses, infertility.8–10 It has been reported that MTX reduces levels of the antioxidant glutathione (GSH) and increases myeloperoxidase (MPO) and malondialdehyde (MDA), 11 the latter of which is an important marker of lipid peroxidation and an indicator of oxidative tissue damage. 12 It has also been reported that MTX causes serious damage to uterine tissue. 13
Coenzyme Q10 (CoQ10) is an oil-soluble component of almost all cell membranes and functions as a diffusible electron carrier in the mitochondrial respiratory chain. 14 It is an important component of cellular energy production in the mitochondria and serves as an antioxidant—scavenging free radicals, decreasing the production of pro-inflammatory cytokines, and inhibiting lipid peroxidation and the oxidation of protein and DNA. 15
Although previous studies have demonstrated that CoQ10 supplementation has a significant protective effect in some tissues, such as the pancreas, muscles, intestines, and brain, there have been insufficient studies on the ovaries and uterus.15–19 The data from existing studies indicates that CoQ10 may be useful for preventing of MTX-induced female reproductive tract injury, and the aim of this study was to determine the possible protective effect of CoQ10 supplementation on MTX-induced utero-ovarian injury in rats.
Methods
Animals
In this study, conducted at the Erzurum Atatürk University Medical Experimental Application and Research Center, 30 female Wistar albino rats (Atatürk University, Erzurum, Turkey) weighing between 250 g and 265 g were kept at temperatures between 21°C and 23°C under a 12 hr light:12 hr dark lighting schedule and fed standard rat chow and water ad libitum. The protocols and procedures were approved by the Atatürk University Animal Experimentation Ethics Committee (Ethics Committee Number: 26.07.2018/152). This study was carried out in accordance with international guidelines on the ethical use of animals.
Chemicals
MTX was obtained from Med-Ilac Turkey (Turkey), thiopental sodium from IE Ulagay (Turkey), and CoQ10 from Solgar (USA).
Experimental groups
The 30 experimental animals were randomly divided into three equal groups, as MTX, MTX+CoQ10 and control (C).
Experimental, surgical, and pharmacological procedures
For the period of the experiment, every morning between 8:00 and 9:00 a.m., each animal cage was carried to the experimental room where a vaginal smear was collected with a plastic pipette filled with 10 µL of normal saline (NaCl 0.9%) by inserting the tip into the rat vagina, but not deeply. Vaginal smear samples taken from each rat were placed on separate slides and examined under a light microscope, without the use of a condenser lens, at 10× and 40× magnification. Three types of cells could be recognized: round and nucleated, which were epithelial cells; irregular without a nucleus, which were cornified cells; and little round cells, which were leukocytes. The proportions of these were used to determine the estrous cycle phases.20,21
For the MTX+CoQ10 (n = 10) group, 10 mg/kg of CoQ10 was orally administered each day; for the MTX (n = 10) and control (n = 10) groups, the same volume of olive oil was orally administered as a solvent. One hour thereafter, 20 mg/kg of MTX was injected intraperitoneally (ip) into the rats of the MTX and MTX+CoQ10 groups. This procedure was repeated daily for a month. At the end of this period, all the rats were euthanized by high dose anesthesia (thiopental 50 mg/kg), and ovary and uterine (midsection of the right uterus horn for each subject) tissues were removed. Malondialdehyde (MDA), myeloperoxidase (MPO), and total glutathione (tGSH) levels were measured in the removed tissues, and histopathological examinations were performed.
Histopathological examination
All tissue samples were first fixed in a 10% formaldehyde solution for light microscope assessment, and the tissue samples were washed under tap water cassettes for 24 hours. The samples were then treated with a conventional alcohol progression (70%, 80%, 90%, and 100%) to remove the water within the tissues, which were then passed through xylol and embedded in paraffin. Sections of 4–5 µm thickness were cut from the paraffin blocks and stained with hematoxylin-eosin. Photos were taken using Olympus DP2-SAL firmware (Olympus® Inc., Tokyo, Japan). Histopathological assessment was performed of all rats by a pathologist who was blind to their group assignment.
Biochemical analysis
Malondialdehyde analysis
MDA measurements were based on the method used by Ohkawa et al., involving spectrophotometrical measurement of the absorbance of the pink-colored complex formed by thiobarbituric acid and MDA. Serum/tissue homogenate samples (0.1 mL) were added to a solution containing 0.2 mL of 80 g/L sodium dodecyl sulfate, 1.5 mL of 200 g/L acetic acid, 1.5 mL of 8 g/L 2-thiobarbiturate, and 0.3 mL of distilled water. The mixture was incubated at 95°C for 1 h. Upon cooling, 5 mL of n-butanol: pyridine (15:1) was added. The mixture was vortexed for 1 min and centrifuged for 30 min at 4,000 rpm. The absorbance of the supernatant was measured at 532 nm. The standard curve was obtained using 1,1,3,3-tetramethoxypropane. 22
MPO activity in serum and tissue
The method defined by Bradley et al. to determine MPO activity in serum/tissue homogenates was used. H2O2 in a phosphate buffer (50 mM, pH 6) was used as a substrate, and an assay buffer was prepared (7.5 mg of o-dianisidine-HCl and 5 mL of 0.0005% H2O2 in 40 mL of phosphate buffer). 20 µL of the serum/tissue homogenate was added to 280 µL of the assay buffer, and the MPO activity was kinetically measured at 460 nm for 5 min. 23
Total glutathione analysis
In the method described by Sedlak and Lindsay, DTNB (5,5’-dithiobis 2-nitrobenzoic acid) disulfide is chromogenic in the medium and is reduced easily by sulfhydryl groups, and the yellow color produced during that reduction is measured by spectrophotometry at 412 nm. For measurement, a cocktail solution (5.85 mL of 100 mM Na-phosphate buffer, 2.8 mL of 1 mM DTNB, 3.75 mL of 1 mM NADPH, and 80 µL of 625 U/L glutathione reductase) was prepared. Before measurement, 0.1 mL of meta-phosphoric acid was added to 0.1 mL of serum/tissue homogenate and centrifuged for 2 min at 2,000 rpm for deproteinization. The 0.15 mL cocktail solution was added to 50 µL of supernatant, and the standard curve was obtained using GSSG. 24
Statistical analysis
SPSS 16.0 was used for the statistical analysis (SPSS Inc., Chicago, IL, USA). Means and standard deviations were calculated, and differences between the three groups were evaluated using ANOVA and Tukey analysis. A value of p < 0.05 was considered to be statistically significant.
Results
The MDA level in the ovarian tissue was 5.8 ± 0.5 µmol/g protein in the control group, rising significantly to 16.5 ± 2.5 µmol/g protein in the MTX group (p < 0.05 relative to the control group). 10 mg/kg/day of CoQ10 reduced the level of MDA significantly to 6.4 ± 0.6 µmol/g protein in the MTX+CoQ10 group (p < 0.05 relative to the MTX group) (Figure 1). Similarly, when compared to the control, MTX caused a statistically significant increase in MPO levels in ovarian tissue (p < 0.05), but CoQ10 reversed this effect, decreasing the MPO levels in the MTX+CoQ10 group significantly (p < 0.05 relative to the MTX group) (Figure 1). Conversely, MTX caused a decrease in tGSH levels in the ovarian tissue of the MTX group rats relative to the control group (p < 0.05), but a dose of 10 mg/kg/day of CoQ10 ameliorated this significantly relative to the MTX group (p < 0.05) (Figure 1). The same pattern of increased MDA and MPO and decreased tGSH in the MTX group relative to the control, all of which were reversed in the MTX+CoQ10 group relative to the MTX group, was also seen in the uterine tissues (p < 0.05 for all comparisons) (Figure 2).
The levels of MDA, MPO and tGSH in ovarian tissues.
MDA, MPO and tGSH levels in uterine tissue.
Histological examination of the ovaries in the control group revealed that the overall ovarian tissue appearance was normal in the cortex and medulla (Figure 3(a)). In the MTX group, microscopic examination showed that there were intensive edema in the follicular and luteal cells; vascular dilatation and congestion; hemorrhage in particular areas; polymorphonuclear cell infiltration; developing follicles that had apparently regressed, with fluid filled cavities; and fewer developing follicles in the ovarian cortex and medulla (Figure 3(b)). In the MTX+CoQ10 group, there was marked amelioration in the general cortex and medulla. Figure 3(c) shows mild vascular congestion, normal follicular structure, and an increased number of developing follicles compared with the MTX group.
Histopathological findings in ovarian and uterine tissues (HE&200). (a) Hematoxylin-eosin staining in ovarian tissue in the C group (DF: developing follicule,
In the control group, all the structures of the uterine tissue were observed to be normal (Figure 3(d)). In the MTX group, the uterine simple cuboidal epithelium showed locally pericellular edema and degeneration. There were mild pericellular edema in the glands, and vascular dilatation and congestion in the mucosa and inflammatory cell infiltration were observed (Figure 3(e). In the MTX+CoQ10 group, the uterine simple cuboidal epithelium showed locally pericellular edema and diminished pericellular edema in the uterine glands, but there was nearly no congestion or inflammatory cell infiltration (Figure 3(f)). Histopathological evaluation was performed in six subjects for each group—one central and five peripheral areas. The degeneration intensity was graded semi-quantitatively as 1, 2, or 3 (mild, moderate, or severe), as shown in Table 1.
Histopathological scoring results of ovarian tissues.
* Compared to SG. ** Compared to MTX group.
As seen in Figure 4, primordial and developing follicles decreased in the MTX group, but CoQ10 significantly increased their numbers. The effect on the atretic follicles and corpus luteum was not statistically significant.
Mean of primordial and developing follicles between groups.
Discussion
In the literature, MTX-induced damage has been reported in different types of tissues,25–27 and high doses of MTX have also been found to have serious toxic effects on ovarian and uterine tissue via a significant decrease in antioxidant levels and increased oxidative stress, causing functional and structural disorders.8,9,28 The accumulation of high levels of reactive oxygen species (ROS) may cause mitochondrial dysfunction, which plays an important role in oxidative stress. 15 Many previous studies evaluating antioxidants as mitochondrial nutrients suggest that abnormalities resulting from mitochondrial dysfunction may be counteracted by the use of antioxidants. 29 For cellular energy production, CoQ10 is an indispensable component for the transport of electrons in the mitochondrial respiratory chain. Moreover, CoQ10 is an antioxidant that restores lipid peroxide levels and the activities of enzymatic and non-enzymatic antioxidants to near normal.30,31
In this study, we investigated the effect of CoQ10 on the harm caused by MTX-induced oxidative stress on ovarian and uterine tissue. MDA is the main product of lipid peroxidation, which is caused by an increase in ROS, 32 and tGSH is the most important indicator of an antioxidant’s capacity to protect the body from the damage of oxidative stress. 33
Previous studies on several different tissue types have shown that oxidative stress causes increased MDA levels and decreased tGSH levels,12,34–36 and the same was found in the MTX group in this study. However, CoQ10 was found to reverse these effects almost to the level of the control group, suggesting that MTX produces oxidative stress in ovarian tissues and that CoQ10 can ameliorate this damage.
We also used MPO as an indicator of inflammation. MPO, which is secreted by activated neutrophils, increases oxygen radicals, which cause oxidative stress and injury. Pınar et al. found increased MPO levels in liver tissue after MTX exposure, 37 and the current study found the same in uterine and ovarian tissue. CoQ10 treatment decreased the MPO levels, suggesting that CoQ10 reversed the inflammatory effect and that the CoQ10-treated group suffered less free radical-induced damage.
Primordial follicles were found to have significantly increased in number in the CoQ10 group, which was also found by Ozcan et al., indicating a potential protective effect of CoQ10 against oxidative stress-related ovarian damage and injury. 15 The histological findings also show that the effect of MTX on the follicle structures was ameliorated in the CoQ10 group, consistent with the biochemical results. Taken together, all these findings demonstrate that CoQ10 is effective in preventing oxidative stress and inflammation induced by MTX in ovary and uterine tissue.
It appears that MTX may cause infertility problems and that CoQ10 protects follicles.
This study has a limitation. Only a single dosage of CoQ10 was examined, and more studies with different doses are needed to determine the mean effective dose.
In conclusion, we demonstrated that MTX leads to oxidative stress-related ovarian and uterine injury, but CoQ10 supplementation protects ovarian and uterine tissue from this effect. CoQ10 may therefore be useful for protecting ovarian and uterine tissue from damage related to MTX, but further studies are required using cells and tissues from human subjects to achieve more accurate results.
Footnotes
Author contributions
Conception and design: Tunay Kiremitli, Sevil Kiremitli. Acquisition of data: Gülce Naz Yazıcı, Mine Gulaboglu. Analysis and interpretation of data: Renad Mammadov. Drafting the article: Betul Kalkan Yilmaz, Ibrahim Hakki Tor. Revising it critically for important intellectual content: Mustafa Burak Akselim. Final approval of the version to be published: Tunay Kiremitli, Sevil Kiremitli. We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors. All authors read and approved the manuscript for publication.
Acknowledgments
We thank Professor Halis Suleyman, Erzincan Binali Yıldırım University Faculty of Medicine, Department of Pharmacology, for his technical support and suggestions.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
