Investigation of Therapeutic Effects of Erdosteine on Polycystic Ovary Syndrome in a Rat Model

• Important treatment options that influence hyperandrogenism and insulin sensitivity are available for PCOS, while long-term treatment options for PCOS are still controversial. This study investigated the therapeutic and protective effects of erdosteine and treatment with metformin in an experimental rat model of PCOS. The results suggest that the therapeutic and protective effect of erdosteine could be due to suppression of biosynthesis of ovarian androgens. Thus, erdosteine may have therapeutic potential in the treatment of PCOS.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age [1]. As far as different diagnostic criteria are concerned, the prevalence of PCOS ranges from 6 to 15% [2]. Though the etiology and pathogenesis of PCOS has not been explained fully, it is characterized by anovulation, hyperandrogenism findings, and abnormal ovarian morphology [3, 4]. Furthermore, PCOS is associated with obesity, metabolic syndrome, hyperinsulinemia, type 2 diabetes, dyslipidemia, and cardiovascular syndrome [5, 6]. Thus, PCOS is an endocrine disorder that presents long-term health risks [7, 8].

Important treatment options that influence hyperandrogenism and insulin sensitivity are available for PCOS, while long-term treatment options for PCOS are still controversial [9]. The aim of treatment of PCOS is to decrease hyperandrogenism and improve insulin sensitivity [9]. Insulin-sensitizers, oral contraceptive pills, and anti-androgenic agents are used commonly for the treatment of PCOS [10, 11]. Treatment with metformin, an insulin sensitizer, increases insulin sensitivity and improves ovulatory function in PCOS [12]. In addition, treatment with metformin improves hyperinsulinemia and menstrual irregularity and reduces androgen levels in women with PCOS [13]. Metformin not only reduces inflammation, it also has an effect on steroidogenesis in ovarian theca cells and granulosa cells [14].

Several agents are used to investigate treatment of PCOS in distinctive experimental and clinical models. Erdosteine [N-(carboxymethylthioacetyl)-homocysteine thiolactone] is a mucoactive agent with antioxidant activity that involves a thiol group [15]. Erdosteine has been reported to protect against hepatic, renal, retinal, and cardiotoxicity [16]. In addition, it is used in clinical practice as a mucolytic drug. Erdosteine is absorbed from the intestine, and after entering the circulation, it is transformed into three metabolites in hepatic circulation. As erdosteine includes thiol groups, it inhibits free radicals present in the circulation. It brings about strong antioxidant effects by increasing the activities of antioxidant enzymes [17].

Although several drugs have been used for the treatment of PCOS, the effectiveness of metformin and erdosteine in PCOS has not been compared. Experimental animal models may help shed light on the pathophysiology of this syndrome. To the best of our knowledge, this is the first study to investigate the therapeutic and protective effect of erdosteine and treatment with metformin in an experimental rat model of PCOS. The main objective of this study was to investigate the potential protective effects of erdosteine in PCOS.

The experimental protocol used in this study was confirmed by the Institutional Animal Care and Ethics Committee of Mustafa Kemal University and performed in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals (publication No. 86-23, revised 1996).

Female adult Wistar albino rats (10 weeks old; weight 275–325 g) were obtained from the Experimental Animal Laboratory of Mustafa Kemal University, Hatay. Rats were caged individually in temperature-controlled steel cages (20–22°C, 55–65% humidity) with a 12-hour light/dark cycle and were provided with standard rat feed without phytoestrogen and water ad libitum. Rats were allowed to acclimatize for 5 days before the experiments. All animals were subjected to daily collection of vaginal secretions for 7 consecutive days in order to evaluate ovarian function; the estrous cycles of rats were evaluated by using vaginal smear method as described by Biswal [18]. Subsequently, only rats with regular estrous cycles were included in the study. Thirty-two rats were randomly classified as four groups with 8 animals in each group. Groups were control group (C), PCOS group, PCOS-metformin treatment group (PCOS+MET), and PCOS-erdosteine treatment group (PCOS+Erd).

Erdosteine (erdosteine suspension) was obtained from Sandoz Pharmaceuticals (Istanbul-Turkey), metformin was obtained from Bilim Pharmaceuticals (Istanbul-Turkey) and letrozole was obtained from Femara-Novartis Pharmaceuticals (Istanbul, Turkey). Ketamine hydrochloride (Ketalar 80 mg/mL) was purchased from Pfizer (Turkey) and xylasine hydrochloride (Rompun 12 mg/mL) was purchased from Bayer (Turkey).

Animal model of PCOS was induced in 24 rats by oral gavage with letrozole once daily at a concentration of 1 mg/kg dissolved in DMSO for 21 consecutive days [19, 20]. Subsequently, the PCOS model evaluation with an anovulatory estrous cycle was verified by analysis of vaginal smear, for which the first one-third of the vaginal wall was taken daily to evaluate the stage of the 4-day ovarian cycle during the examination of the study period. Following the induction of PCOS, 8 rats with PCOS were included in the PCOS group and did not receive any treatment. Rats in the PCOS+MET group (n = 8) were treated orally with 500 mg/kg/day of metformin, while rats in the PCOS+Erd group (n = 8) were treated orally with 100 mg/kg/day of erdosteine for 30 days [15]. Animals in the control group received 1 mL/kg/day of saline solution orally for 30 days.

All animals were weighed at the beginning and end of study. The rats were anesthetized with a single intraperitoneal injection of ketamine (80 mg/kg) and xylasine (12 mg/kg). The blood samples were obtained by cardiac puncture and transferred to EDTA-containing tubes. Plasma was separated by cold-centrifugation (4°C) at 3,000 rpm for 10 min. Ovarian tissues were excised for histological analysis and fixed in 10% formaldehyde (PBS 10 mM, pH 7.4). Plasma samples were stored in a freezer at –80°C for biochemical analysis.

Ovaries and uteruses of rats were removed and weighed prior to histological examination. After cutting through at the longest lengthwise dimension, ovarian tissues were fixed in 10% neutral formalin for 24 h. Then all samples were cleaned, dehydrated, and embedded in paraffin. Four-millimeter-thick sections were prepared using a microtome (Leica RM 2145 RTS, Nussloch, Germany) and stained with hematoxylin and eosin. A light microscope (Olympus Clinical Microscope BX53, Tokyo, Japan) was used to evaluate all samples according to the methods of Ergenoglu et al. [21] and Atis et al. [22]. All histological slides were examined in a blinded fashion by an experienced pathologist. Follicles containing an oocyte with a nucleus were counted and described as healthy. Follicles were grouped according to the following definitions:

• Preantral follicle: a follicle with an intact, enlarged oocyte with a visible nucleus and a single layer of cuboidal granulosa cells.

• Antral follicle: a follicle with two or more layers of cuboidal granulosa cells, whether the cavity was apparent or not.

• Atretic follicle: a follicle containing a degenerating ovum or pyknotic granulosa cells.

• Cystic follicle: a follicle that is a large fluid-filled structure with an attenuated granulosa cell layer and a thickened theca internal cell layer.

For each group, the number of corpus luteum, preantral, antral, atretic, and cystic follicles of ovaries were evaluated and counted. The thickness of the tunica albuginea, hyperplasia of the theca internal cells, reduction in corpus luteum, and sub-capsular follicular cysts in the control group and PCOS groups were analyzed (Fig. 1).

Serum total cholesterol (Total-C), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG) were measured using an automated clinical chemistry analyzer (Abbott diagnostic, Architect C 8000, Abbott Park, IL, USA). Serum insulin levels were measured using a chemiluminescence method (Siemens, Immulite 2000 XPI, Malvern, PA, USA). Insulin resistance was measured with a homeostasis model assessment for insulin resistance (HOMA-IR) calculated as previously [23]. Serum levels of estrone (E1), estradiol (E2), testosterone (TT), and androstenedione (AS) were determined using a competitive enzyme-linked immunosorbent assay [24] using commercially available ELISA kits (Awareness Technology Inc., Chromate ELISA Reader, Palm City, FL, USA).

Serum samples for the measurement of total antioxidant status (TAS) and total oxidant status (TOS) were stored at –80°C until needed. The TOS and TAS of the rat serum samples were measured automatically with an Architect C8000-Abbott auto-analyzer device (Abbott, Wiesbaden, Germany) at the biochemistry laboratory of MKU Hospital. The oxidative stress index (OSI) was calculated by the formula TOS (μmol H₂O₂ equivalent/L)/TAS (μmol Trolox equivalent/L).

Oxidants present in a sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by abundant glycerol molecules in the reaction medium. The ferric ion forms a colored complex with xylenol orange in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules in the sample. The assay was calibrated with hydrogen peroxide. The results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (μmol H₂O₂ Eq/L).

This was done by the new automated measurement technique described by Erel [25]. This technique measures the antioxidant effect of the sample against the potent free radical reactions initiated by hydroxyl radicals. The assay had sensitivity of < 3%. The findings are expressed as mmol Trolox Eq/L [25].

The normality of the distribution of variables was analyzed by the Kolmogorov-Smirnov normality test. Normally distributed data was analyzed by one-way analysis of variance, and also nonparametric variables between different groups were compared using Kruskal-Wallis test. The data were analyzed by one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. Also, Wilcoxon test was used for comparison between initial and last rat weights. All values were reported as mean ± SEM. Statistical significance was considered to be p < 0.05. The analyses were performed using Graph Pad Prism software (La Jolla, CA, USA).

The average value of uterine and ovarian weights and histopathological parameters is shown in Table 1 and Figure 1. Histopathological analysis of PCOS-only ovaries appeared to be substantially increasing in subcapsular cyst formation, capsular thickness, and theca cell hyperplasia in addition to a reduced corpus luteum. There was no difference between groups in terms of average of preantral and antral follicle counts, despite the fact that preantral follicles were statistically lower in the PCOS+Erd group as compared to the PCOS+MET group (p < 0.05), and antral follicles seem to be higher in the PCOS+Erd group when compared to the PCOS group (Table 1). Statistically significant differences between groups were seen in terms of average counts of atretic and cystic follicles (Table 1). The cystic follicle count of the PCOS group was significantly higher when compared with the control, PCOS+MET, and PCOS+Erd groups (p < 0.01, p < 0.01, and p < 0.01, respectively). The atretic follicle counts were significantly lower in the control, PCOS+MET, and PCOS+Erd groups when compared with the PCOS group (p < 0.05, p < 0.01, and p < 0.001, respectively). There was no significant difference between the PCOS+MET and PCOS+Erd group with respect to numbers of cystic and atretic follicles (p > 0.05). Corpus luteum numbers were lower in the PCOS group as compared with the control, PCOS+MET, and PCOS+Erd groups (p < 0.05, p < 0.001, and p < 0.01, respectively). There was no significant difference between the PCOS+MET and PCOS+Erd groups with respect to corpus luteum number (p > 0.05).

Biochemical parameters and weights of rats are presented in Table 2. The final weights of rats in the PCOS, PCOS+MET, and PCOS+Erd groups were statistically higher than their initial weights (p < 0.001, p < 0.05, and p < 0.05, respectively). Fasting glucose levels were higher in the PCOS group than in the control and metformin groups (p < 0.001 and p < 0.05, respectively). There was no significant difference in the PCOS+Erd group as compared to the control and PCOS groups. HOMA-IR was substantially higher in the PCOS group as compared with the control, PCOS+MET, and PCOS+Erd groups (p < 0.001, p < 0.01, and p < 0.001, respectively). However, there were no substantial differences in HOMA-IR levels between the PCOS+MET and PCOS+Erd groups. Total cholesterol levels were higher in the PCOS group as compared with the control and PCOS+MET groups (p < 0.01 and p < 0.05, respectively). Triglyceride levels were also substantially higher in the PCOS group as compared to the control, PCOS+MET, and PCOS+Erd groups (p < 0.05, p < 0.05, and p < 0.05, respectively). LDL-C levels were statistically lower in the control, PCOS+MET, and PCOS+Erd groups as compared to the PCOS group (p < 0.01, p < 0.05, and p < 0.01, respectively). On the other hand, HDL-C levels were lower in the PCOS group as compared to the PCOS+MET and PCOS+Erd groups (p < 0.05 and p < 0.05, respectively). Levels of TAS were not substantially different between groups. TOS was statistically lower in the control, PCOS+MET, and PCOS+Erd groups as compared to the PCOS group (p < 0.0001, p < 0.05, and p < 0.01, respectively). Furthermore, OSI was statistically lower in the control, PCOS+MET, and PCOS+Erd groups as compared to the PCOS group (p < 0.0001, p < 0.01, and p < 0.0001, respectively). There was a significant difference between the PCOS+MET and PCOS+Erd group with respect to OSI (p < 0.05).

A statistically significant reduction was seen in levels of estrone in the control, PCOS+MET, and PCOS+Erd groups as compared to the PCOS group (p < 0.001, p < 0.05, and p < 0.001, respectively); estradiol levels were lower in the PCOS group as compared to the control, PCOS+MET, and PCOS+Erd groups (p < 0.01, p < 0.001, and p < 0.001, respectively). The E1/E2 ratio was significantly higher in the PCOS group as compared to the control, PCOS+MET, and PCOS+Erd groups (p < 0.001, p < 0.001, and p < 0.001, respectively). Likewise, levels of testosterone and androstenedione were substantially higher in the PCOS group as compared to the control, PCOS+MET, and PCOS+Erd groups (Table 3).

This study shows that erdosteine ameliorates lipid profile, insulin resistance, hormone profiles, and follicle counts in PCOS. Women with PCOS are in need of long-term treatment because PCOS is a lifelong endocrinopathy. Even though several drugs have been used to treat PCOS, the effectiveness of erdosteine in PCOS has not been investigated. Previous experimental studies have shown that erdosteine could be used at a wide range of dose without any side effects; however, metformin induces many side effects after long-term use.

Metformin and oral contraceptive pills have been used to treat the symptoms experienced by women with PCOS [13]. When metformin is administered as an insulin sensitizer, substantial reductions in serum insulin levels and insulin resistance are observed and it also improves ovulatory functions. Although metformin and oral contraceptive pills have been beneficial in treating PCOS symptoms, their efficacy and safety are still not entirely elucidated [13, 26]. The observed effects of drugs used for PCOS are usually not significant enough for treatment. Furthermore, previous studies have indicated that metformin produces many side effects after prolonged usage [27]. These observations made us interested in exploring new therapeutic options for the therapy of PCOS.

Erdosteine is associated with a low incidence of adverse events, most of which are gastrointestinal and generally mild. The LD₅₀ (median lethal dose) is very high, 3,500–5,000 mg/kg [28]. PCOS is a low-grade chronic inflammatory oxidative state [29]. In this study, we compared the effects of erdosteine and metformin. There was a significant reduction in the numbers of atretic and cystic follicles in both metformin and erdosteine groups. Furthermore, there was a substantial reduction in the number of preantral follicles in the erdosteine group as compared to the metformin group, probably due to its antioxidant effects. Our histological analysis indicates that erdosteine may benefit follicular development in the rat model of PCOS.

There was a substantial difference in ovarian and uterine weights between groups; while uterine weights were low, ovarian weights were high in the PCOS group. Similar findings regarding ovarian and uterine weights were reported in letrozole-induced PCOS models [19]. Uterine weights in the metformin and erdosteine groups are higher than in the PCOS group, probably due to an increase in estrogen levels.

In addition to morphological improvements in the ovaries, we observed that levels of total testosterone in the metformin and erdosteine groups were significantly lower than in the PCOS group. There have been different findings on E1, E2, and androstenedione concentrations in letrozole-induced PCOS rat models [30]. In our study, it appears that erdosteine substantially affected levels of E1, E2, and androstenedione. These findings may be considered as a reflection of histopathological amelioration brought about by erdosteine. However, the histological data does not reveal the mechanism by which erdosteine decreases the appearance of PCOS in ovaries. Erdosteine may affect androgen levels. On the other hand, it seems that the precise effect of metformin on androgen levels compared to the erdosteine treatment group was not studied. This may be due to the possible effect of metformin on the reduction of androgens by regulating insulin resistance [31].

PCOS has been shown to be associated with several forms of dyslipidemia with low levels of HDL-C, high levels of triglycerides, total cholesterol, and LDL-C [24]. In our study, levels of HDL-C were significantly higher in metformin-treated and erdosteine-treated animals than in untreated rats with PCOS. Furthermore, levels of triglycerides, total cholesterol, and LDL-C were significantly lower in metformin-treated and erdosteine-treated rats than in the PCOS group.

The etiology of insulin resistance in PCOS is still not clear. Insulin resistance was described as HOMA-IR [32]. In our study, we observed that HOMA-IR is significantly lower in the PCOS+MET and PCOS+Erd groups compared to the PCOS group. Glucose also is significantly lower in the PCOS+MET group as compared to the untreated PCOS group. In previous studies, it was indicated that hypovitaminosis D may contribute to worsen insulin resistance, which is a feature of women with PCOS [33]. Therefore, vitamin D replacement therapy is given to PCOS patients. In terms of the same effect of vitamin D, erdosteine may have a beneficial effect on insulin resistance in women with PCOS [34].

We suggest that these clinical results are associated with the antibacterial, antioxidant, and mucolytic effects of erdosteine [35, 36]. The positive effect of erdosteine in PCOS is likely to be associated with the thiol groups in its structure. Erdosteine has been used to ameliorate ischemia reperfusion injury in several organs [15]. Erdosteine may also protect against ovarian ischemia/reperfusion with the release of oxidant free radicals and prevention of pro-inflammatory processes [15]. Also, erdosteine, due to its antioxidant effects, may reduce the adverse effects caused by increased levels of reactive oxygen species (ROS). We chose to test erdosteine because of its antioxidant effects, because it has been used successfully in clinical and experimental studies, and because it is cheap and is easily available. Taken together, our analysis supports a protective role of erdosteine in a rat model of letrozole-induced PCOS.

This study shows that erdosteine treatment increases the numbers of antral follicle and corpus luteum, and reduces the numbers of atretic and cystic follicles. In addition, it decreases levels of total testosterone, E2, and androstenedione. This study also shows that the therapeutic effect of erdosteine could be due to suppression of biosynthesis of ovarian androgens. Thus, erdosteine may have therapeutic potential in the treatment of PCOS.

The authors have no conflicts of interest to disclose.

A.K. and R.D. designed and conducted the study; R.D. provided the rats for the experiments. H.D., O.T., and G.A. performed the application of treatment agents. O.T., H.D., R.D., and G.A. performed together surgical technique. R.D. and A.K. wrote the manuscript. C.T. provided supervision. O.T. and Z.A.T. evaluated histological analysis. All of the authors participated in the interpretation of the studies and analysis and also approved the final version of the manuscript for submission.