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Effects of resveratrol on doxorubicin induced testicular damage in rats夽 Sibel Türedi a,∗ , Esin Yulu˘g a , Ahmet Alver b , Ömer Kutlu c , Cemil Kahraman b a b c
Karadeniz Technical University, Faculty of Medicine, Department of Histology and Embryology, 61080 Trabzon, Turkey Karadeniz Technical University, Faculty of Medicine, Department of Medical Biochemistry, 61080 Trabzon, Turkey Karadeniz Technical University, Faculty of Medicine, Department of Urology, 61080 Trabzon, Turkey
a r t i c l e
i n f o
Article history: Received 12 March 2014 Accepted 8 December 2014 Keywords: Doxorubicin Testicular toxicity Apoptosis Resveratrol Infertility
a b s t r a c t The purpose of this study was to evaluate the likely protective effect of resveratrol (RES) on doxorubicin (DOX) induced testicular damage. Rats were divided into five groups: control, RES, dimethyl sulfoxide (DMSO), DOX and DOX + RES. At the end of treatment, the rats were sacrificed. Plasma testosterone levels, oxidative status, epididymal sperm parameters and testicular apoptosis were evaluated. MDA levels, GPx and GSH activities were higher in the DOX group than in the control group. MDA levels were lower in the DOX + RES group than in the DOX group. The DOX group exhibited a significant decrease in plasma testosterone levels, sperm concentration and motility, and a significant increase in abnormal sperm rate and TUNEL (+) cells in the testis. A significant increase was observed in plasma testosterone levels and sperm concentration and motility, and a significant decrease in the abnormal sperm rate and TUNEL (+) cells in the DOX + RES group compared to the DOX group. A marked improvement in severe degenerative alterations in the germinative epithelium was also observed following treatment with RES. In conclusion, RES makes a positive contribution to fertility by exhibiting anti-apoptotic and antiperoxidative effects against DOX-induced testicular damage. © 2014 Published by Elsevier GmbH.
1. Introduction Anticancer therapy generally compromises physiological hemostasis and causes multi-organ failure during the therapeutic process (Ayla et al., 2011). Doxorubicin (DOX) is an antracyclinegroup antibiotic that has been used as an antineoplastic agent in the treatment of various hematological malignities, including solid tumors, for many years (Doroshow et al., 1980; Gharanei et al., 2013). Due to its severe side-effects on the testis, as well as toxicity in various organs (Yeh et al., 2009), its clinical use is limited, however (Miranda et al., 2003; Ayla et al., 2011). DOX treatment may damage male fertility by adversely affecting sperm development, production and count (Prahalathan et al., 2005a). Previous studies have shown that it causes apoptosis in the seminiferous epithelium cycle (Sjoblom et al., 1998) and chromosomal damage in germ cells (Au and Hsu, 1980). The exact mechanism responsible
夽 The abstract of this study was presented at the 2012 XIth National Histology & Embryology Congress in Turkey (with international contribution). ∗ Corresponding author. Tel.: +90 462 3777736; fax: +90 462 3252270. E-mail addresses: [email protected], [email protected] (S. Türedi), [email protected] (E. Yulu˘g), [email protected] (A. Alver), [email protected] (Ö. Kutlu), [email protected] (C. Kahraman).
for DOX’s testicular toxicity is unclear, although recent findings point to oxidative stress and cellular apoptosis as major causes (Prahalathan et al., 2004; Yeh et al., 2009). DOX therapy-related oxidative damage is mediated by an oxyradical complex containing superoxide, hydroxyl radical and a certain amount of iron (Hida et al., 1995). Oxyradicals lead to damage in the mitochondrial and other cytoplasmic organelle membranes through proteins, nucleotides and phospholipids (Muraoka and Miura, 2003). Resveratrol (RES) (trans-3,4 ,5-trihidroksi-stilben) is a natural phytoalexin found in various plants, such as grape, peanut and mulberry (Krasnow and Murphy, 2004; Caruso et al., 2004). RES’ biological activities are known to exhibit antioxidant, antiinflammatory, anticancer, antiatherogenic and cardioprotective properties (Ignatowicz and Baer-Dubowska, 2001; Dudka et al., 2012; Benayahoum et al., 2013). In addition, RES’ antioxidant effect may also help protect against DNA damage occurring after a rise in ROS levels and against lipid peroxidation in the cell membrane (Jiang et al., 2008). Studies have shown that it is more effective that vitamins E and C, the best known antioxidants (Belguendouz et al., 1998). In another study, grapeseed extracts containing RES were shown to protect glial cells against oxidative stress (Royshowdhury et al., 2001). The antioxidant activity of RES is associated with its ability to inhibit ribonucleotide reductase and cyclooxygenase transcription in DNA polymerase activity (Frémont, 2000).
http://dx.doi.org/10.1016/j.etp.2014.12.002 0940-2993/© 2014 Published by Elsevier GmbH.
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The purpose of this study was to evaluate the likely protective effect of RES on DOX-induced testicular damage using histopathological and biochemical analyses. 2. Materials and methods 2.1. Animals and experimental design Thirty male Sprague–Dawley rats (5 weeks old, weighing 140–170 g) were used. All animals were kept in standard laboratory conditions. Standard laboratory chow and water were provided ad libitum throughout the experiment. The study was approved by the Institutional Animal Ethical Committee of Karadeniz Technical University, Trabzon, Turkey. The rats were randomly divided into five groups. The control group (n: 6) was given a single dose of 9% isotonic sodium chloride intraperitoneally (i.p.). The RES group (n: 6) was given 20 mg/kg RES i.p. (R5010-500 mg, Sigma–Aldrich, St. Louis, MO, USA) per day [dissolved in 30% dimethyl sulfoxide (DMSO)] every day throughout the experiment, while the DMSO group (n: 6) was administered 30% DMSO solution i.p. The DOX group (n: 6) was injected with a single dose of 10 mg/kg DOX (Adriblastina 10 mg, DEVA, Turkey) i.p. The DOX + RES group (n:6) was administered a single dose of 10 mg/kg DOX together with 20 mg/kg RES i.p. per day throughout the experiment. Dosages of RES (Oktem et al., 2012) and DOX (Atessahin et al., 2006) were determined on the basis of reports from previous studies. 2.2. Sample collection At the end of the 21-day experiment, rats were sacrificed by decapitation under ketamine (Ketalar, Pfizer, Turkey) anesthesia. The abdominal cavity was quickly opened and the testes extracted and separated from the epididymises. The right testicular epididymis was placed in a Petri dish containing 2 mL Tris buffer solution for sperm parameter analysis. The right testis and the left epididymis were fixed in Bouin’s solution for histological analysis. Blood and tissue specimens collected for biochemical parameters were stored at −80 ◦ C. 2.3. Biochemical analysis Plasma malondialdehyde (MDA) levels were determined using the method described by Yagi (1994). Tetramethoxypropane was used as a standard, and MDA levels were calculated as nmol/mL. Tissue MDA levels in testis samples were measured using the method described by Uchiyama and Mihara (1978). Tetramethoxypropane was used as a standard, and MDA levels were calculated as nanomoles per gram wet tissue. Superoxide dismutase (SOD) and catalase (CAT) activities were determined in the remaining part of the testis tissue. SOD activities were measured using the method described by Sun et al. (1988). Enzyme activity leading to 50% inhibition was taken as one unit, and the results were expressed as U/mg protein. CAT activity was determined using the method described by Aebi (1974), and the results were expressed as k/g protein (k, rate constant). Protein concentrations were determined following Lowrey’s method (1951). Glutathione peroxidase (GP-x) activity was determined using a commercial spectrophotometric kit (Cayman, USA). Glutathione (GSH) levels were analyzed using high-performance liquid chromatography (Afzal et al., 2002) (Agilent 1100 series HPLC and fluorescence detector systems, Waldbronn, Germany; column, Fortis UniverSil C18, 4.6 mm × 250 mm). Results were expressed as g/mL. Tissue glucose-6-phosphate dehydrogenase (G6PD) and sorbitol dehydrogenase (SDH) activities were measured using the methods described by Deutsch (Aebi, 1986a) and Gerlach (Aebi,
1986b), respectively. The results were expressed as U/g protein. Plasma testosterone levels were measured using a rat testosterone EIA kit (Cayman, USA).
2.4. Histopathological examinations Testis and epididymis specimens were fixed, embedded in paraffin blocks, sliced into 5-m sections and stained with hematoxylin–eosin (H&E) for histopathological evaluation. All testicular histology was examined by a histologist under light microscopy (Olympus BX-51; Olympus, Tokyo, Japan). For seminiferous tubule diameter (STD) and germinal epithelium thickness (GET) measurement, 10 different areas were selected at random from each testis section. The Analysis 5 Research program (Olympus Soft Imaging Solutions, Germany) was used under light microscopy at a magnification of 200×, and 20 seminiferous tubules were selected and measured on each testis section. Johnsen’s tubular biopsy score (JTBS) was used to evaluate spermatogenesis. Under this system, testis seminiferous tubule sections were scored from 0 to 10 (Johnsen, 1970).
2.5. TUNEL assay Testicular apoptosis was analyzed using the terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate nick end labeling assay (TUNEL) method. Sections 5 m in thickness were taken from the paraffin blocks and subjected to standard deparaffinization. TUNEL staining of sections was performed using an in situ cell death detection kit AP kit (Roche, Mannheim, Germany), in accordance with the manufacturer’s instructions. The proportion of TUNEL (+) spermatogenetic cells to the number of normal spermatogenetic cells was taken as the testis apoptotic index (TAI) (Karaguzel et al., 2012).
2.6. Epididymal sperm concentration, motility and abnormal sperm rate The epididymis was broken down in a Petri dish containing 2 mL Tris buffer tampon solution and incubated for 30 min at 37 ◦ C for the sperm to swim in the fluid. Counts, motility and abnormal sperm levels were calculated. A Makler sperm counting chamber (Sefi-Medical Instrument, Haifa, Israel) was used to calculate sperm concentrations. Briefly, 5 L of semen from the homogenate was dropped into the center of the chamber and the glass lid was closed. Sperm were made to swim at a depth of 10 m with the assistance of four quartz blades. Sperm count was then calculated under a light microscope at 200× magnification (%106 ). Sperm motility was calculated by modifying the method described by Sonmez et al. (2005). One drop of sperm fluid diluted in Tris buffer solution was collected by pipette, placed onto a slide and covered with a slide cover. Sperms were evaluated as motile or immotile under a light microscope at 400× magnification. Three different areas were selected for each specimen, and the mean of these was taken as the motility score. Sperm morphology was calculated by modifying the method described by Trivedi et al. (2011). Sperm containing solution was centrifuged at 1000 rpm for 3 min. Subsequently, 2–3 drops of solution were spread on a slide, dried, fixed with methanol for 3 min and stained with 1% eosin-y. Two hundred sperm from each animal were examined for head and tail anomalies under a light microscope under immersion oil at a magnification of 100×, and were classified as normal, head abnormal or tail abnormal (Xin et al., 2012).
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Table 1 Clinical signs, seminiferous tubule diameter, germinal epithelium thickness, testis histopathologic damage scores and apoptotic index in all groups. Parameters
Control (n:6)
Body weight (g) Testis weight (g) STD GET JTBS TAI (%)
212.8 1.43 259 90 9.73 2.74
± ± ± ± ± ±
RES (n:6)
16.76 0.17 4.37 4.47 0.37 1.13
189.5 1.25 238.3 87.5 9.76 3.11
± ± ± ± ± ±
DMSO (n:6)
12.53 0.08 4.72 2.42 0.15 1.02
189.6 1.25 241 86.57 9.63 3.04
± ± ± ± ± ±
DOX (n:6)
15.31 0.1 9.1 3.34 0.18 0.87
150.1 0.61 151.4 49.02 5.68 56.16
± ± ± ± ± ±
DOX + RES (n:6)
16.72a 0.13a 14.6a 6.80a 0.62a 5.11a
159.2 1.04 224 82.87 8.97 20.44
± ± ± ± ± ±
13.96a 0.14a , b 4.72b 2.57b 0.48b 2.65a , b
The data represent the mean ± SD. a P < 0.05 compared with control. b P < 0.05 compared with DOX group.
Table 2 Oxidant–antioxidant biochemical parameteres and plasma testosterone levels in all groups. Parameters Plasma testosterone (Pg/mL) Plasma MDA (nmol/L) Tissue MDA (nmol/g) GP-x (U/mg protein) GSH (g/mL) SOD (U/g tissue) CAT (k/g)
Control (n:6) 1025 0.68 377.4 1.43 4.19 6.66 0.02
± ± ± ± ± ± ±
288.5 0.19 44.35 0.16 0.07 1.77 0.03
RES (n:6) 821.6 0.74 385.7 1.39 4.41 5.96 0.01
± ± ± ± ± ± ±
36.3 0.44 42.61 0.42 0.33 1.41 0.002
DMSO (n:6) 848.9 0.62 347 1.67 4.18 5.80 0.01
± ± ± ± ± ± ±
99.7 0.19 62.58 0.49 0.19 0.82 0.002
DOX (n:6) a
428.5 1.30a 503a 2.01a 5.86a 5.07 0.007
± ± ± ± ± ± ±
129.8 0.48 27.45 0.34 0.33 1.82 0.001
DOX + RES (n:6) 800.5b 0.85b 448.3b 2.19a , b 6.31a 5.26 0.10
± ± ± ± ± ± ±
261.6 0.10 48.23 0.26 0.71 1.56 0.002
The data represent the mean ± SD. a P < 0.05 compared with control. b P < 0.05 compared with DOX group.
2.7. Statistical analysis All statistical analyses were performed using computer software (Prism 5.0, Graphpad Software Inc., San Diego, CA, USA). Data were presented as mean (±) standard deviation (SD). Kruskal–Wallis analysis of variance and the Mann–Whitney U-test with Bonferroni correction were used for statistical analysis. Significance was set at P < 0.05.
3. Results 3.1. Clinical signs Body weight in the DOX group decreased significantly compared to the control group (P < 0.05). Although an increase was observed in the DOX + RES group compared to the DOX group, this was not significant (Table 1). Testis weight in the DOX group decreased significantly compared to the control group. DOX + RES group values were significantly higher compared to those of the DOX group (P < 0.05) (Table 1).
3.2. Biochemical analysis results Experimental group plasma testosterone, plasma MDA and tissue MDA levels and SOD, CAT, GP-x and GSH activity findings are shown in Table 2. Plasma testosterone levels decreased significantly in the DOX group compared to the control group. A significant increase was observed in the DOX + RES group compared to the DOX group (P < 0.05). Plasma and tissue MDA levels and GP-x and GSH activities increased significantly in the DOX group compared to the control group. Plasma and tissue MDA levels decreased significantly in the DOX + RES group compared to the DOX group (P < 0.05), while GP-x activity rose significantly. No significant difference was determined in testicular tissue SOD and CAT activities between the groups (P > 0.05). In addition, although a decrease was observed in G6PD and SDH enzyme levels in the DOX group (0.09 ± 0.66 and 2.65 ± 1.19, respectively)
compared to the control group (0.19 ± 0.05 and3.49 ± 2.05, respectively), this was not significant. 3.3. Histopathological findings Testis and epididymis tissues in the control (Fig. 1A and D), RES and DMSO groups had a normal histological appearance. Disorganization in the seminiferous tubule basal membrane and seminiferous epithelium, vacuolization, a decrease in germinal cells, and unmatured germinal epithelial cells in the lumen were observed in the DOX group (Fig. 1B). Few spermatozoa were seen in the lumen in the epididymal tissue of the DOX group (Fig. 1E). In the DOX + RES group, although there were a few tubules in which non-matured germinal epithelial cells were observed in the lumen (Fig. 1C and F), the appearance was close to that of the control group. In the DOX group, STD and GET decreased significantly compared to the control group (P < 0.05). A significant increase was observed in the DOX + RES group compared with the DOX group (P < 0.05) (Table 1). JTBS in the DOX group decreased significantly compared to the control group (P < 0.05). A significant increase was observed in the DOX + RES group compared to the DOX group (P < 0.05) (Table 1). TAI rose significantly in the DOX group compared to the control group. A significant decrease was seen in the DOX + RES group compared to the DOX group (P < 0.05) (Table 1). TUNEL (+) cells in the DOX group were widespread in the germinal epithelial cells of the seminiferous tubule (Fig. 2B). There was a significant decrease in TUNEL (+) cells in the DOX + RES group (Fig. 2C). 3.4. Epididymal sperm concentration, motility and abnormal sperm rate Sperm concentration, motility and abnormal sperm rate results are shown in Table 3. Following DOX administration, a significant decrease in sperm concentration and motility and a significant increase in abnormal sperm numbers were observed in comparison to the control group (P < 0.05). A significant increase in sperm concentration and motility, and a significant decrease in abnormal
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Fig. 1. Testis and epididymal section micrographs: (A, D: control group; B, E: DOX group; C, F: DOX + RES group): (A) normal seminiferous tubule germinal epithelium (arrow) and spermatozoa in the lumen (arrowhead); (B) vacuoliation and degeneration in the germinal epithelium (arrow), decreased spermatozoa (arrowhead) and germinal cell clustering in the lumen (stars); (C) nearly normal seminiferous tubule germinal epithelium (arrow), very few immature germinal cells (star) and spermatozoa (arrowhead) in the lumen (H&E 200×); (D) normal epididymal histology; (E) decreased spermatozoa (stars) and spermatids clustering in the epididymal lumen (arrows); (F) spermatozoa (arrowhead) and very few spermatids (arrows) in the epididymal lumen (H&E 400×).
Fig. 2. Testis seminiferous tubule sections stained with the TUNEL technique (original magnification 400×) (A: control group; B: DOX group; C: DOX + RES group). Apoptotic spermatogenetic cells (arrows).
Table 3 Epididymal sperm concentration, motility, abnormal sperm rate in all groups. Parameters
Control (n:6)
RES (n:6)
DMSO (n:6)
Sperm concentration (×106 ) Motility (%) Abnormal sperm rate (%)
22.83 ± 6.52 73.1 ± 9.23 2.48 ± 0.09
20.17 ± 0.75 68.34 ± 9.86 2.31 ± 0.20
19.67 ± 1.5 71.53 ± 8.99 2.21 ± 0.14
DOX (n:6) 4.5a ± 3.50 8.10a ± 15.07 5.80a ± 0.85
DOX + RES (n:6) 15.14a , b ± 3.97 57.86b ± 6.46 3.89b ± 0.83
The data represent the mean ± SD. a P < 0.05 compared with control. b P < 0.05 compared with DOX group.
sperm count were observed in the DOX + RES group compared to the DOX group (P < 0.05).
4. Discussion Testicular dysfunction is one of the serious side-effects of cytotoxic chemotherapy (Prahalathan et al., 2005b). DOX is a widely used cytotoxic agent known to impair testicular function and spermatogenesis (Ceribasi et al., 2012). Protection of fertility is important during the use of chemotherapeutics. The present study is an attempt to evaluate the role of resveratrol in DOX-induced testicular dysfunction in rats. In our study, testis weight decreased significantly in the DOX administered group. Kato et al. (2001) observed a decreased in testis weight in chronic exposure to DOX. We think that this decrease after DOX administration may be due to severe parenchymal atrophy in the testicular seminiferous tubule (Sudha and Kavimani, 2011).
ROS species such as superoxide ions, hydroxyl radical, peroxyl and alkoxy radicals and the non-radical oxygen, hydrogen peroxide and ozone are produced during oxygen use in normal metabolism, and are required for various physiological conditions in the male reproductive system. However, excessive production of ROS leads to lipid peroxidation, and this results in oxidative stress. Cells employ various antioxidant mechanisms to reduce the levels of excess ROS. Antioxidant enzymes such as SOD and CAT react against radicals such as superoxide and hydrogen peroxide, while GP-x has a scavenging effect against alkyl, alkoxy and peroxyl radicals that can form from oxidized membrane compounds, and uses GSH as a substrate (Ceribasi et al., 2012). Previous studies have reported that oxidative stress is the mechanism responsible for toxicity developing in testicular tissue due to DOX (Lebrecht et al., 2007; Trivedi et al., 2011). In present study, a significant increase in MDA levels was observed in the DOX group, indicating increased lipid peroxidation (Ceribasi et al., 2012). This suggests that the increase may be due to DOX-associated excessive ROS production and a rise in lipid
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peroxidation. GSH plays an important role in the detoxification of free radicals and of ROS’ antioxidative and xenobiotic compounds (Ayla et al., 2011). Salvemini et al. (1999) reported that GSH synthesis may develop as an adaptation process in situations in which cells are exposed to oxidative stress. Similarly, Yilmaz et al. (2006) confirmed that GSH synthesis may be a positive regulation under oxidative stress resulting in a rise in GSH concentrations. GP-x and GSH activities also rose significantly in the DOX group compared to the control group in our study. Although a decrease was seen in SOD and CAT activities, this was not statistically significant. The increase in GP-x and GSH activities in this study may be attributed to excess production in order to scavenge free radicals produced in excess quantities under oxidative stress (Ceribasi et al., 2012). SDH is involved in the provision of energy for sperm cells and converts sorbitol and fructose. A decrease in the activity of this enzyme after DOX treatment compromises the distribution of energy metabolism in sperm cells. Another key enzyme in testicular tissue is G6PD, which gives rise to an equivalent decrease for the hydroxylation of steroids. G6PD is directly involved in glutathione metabolism (Das et al., 2012). Low levels of this enzyme increase oxidative stress and may lead to cell death. Prahalathan et al. (2004) reported lower levels of this enzyme in a group treated with DOX compared to a control group. In our study, while G6PD and SDH enzymes were lower in the DOX group compared to the control group, the difference was not significant. The changes observed after DOX administration in a previous study indicated serious oligospermia in the epididymis after 4 weeks and azoospermia after 9 weeks (Zanetti et al., 2007). In this study, sperm concentration and motility decreased significantly and the abnormal sperm rate rose significantly in the DOX group. Another previous study demonstrated that 10 mg/kg−1 DOX treatment led to a decreased in motility, but not in sperm concentration (Atessahin et al., 2006). Spermatozoa plasma membranes contain large amounts of saturated fatty acids, and antioxidant enzyme levels in their cytoplasm are very low. Oxidative stress, which exhibits high sensitivity for denaturation and fragmentation, may play a significant role in the emergence of sperm anomalies by affecting sperm DNA (Sikka, 1996). Previous studies have shown that DOX causes DNA fragmentation and chromosome damage (Menna et al., 2010; Xin et al., 2012) and that increasing oxidative stress causes a decrease in the number of motile sperm and a rise in abnormal sperm rates (Atessahin et al., 2006). Testosterone is needed for spermatogenesis, the establishment of structural morphology and the physiology of the seminiferous tubule (Das et al., 2012). Juan et al. (2005) observed an increase in serum testosterone and gonadotropin levels, a decrease in tubular density and a significant increase in sperm numbers of rats given resveratrol at 20 mg/kg per day over 90 days. In our study, there was a significant increase in sperm maturation and plasma testosterone levels in rats given DOX and concomitant resveratrol compared to the DOX group. This may be due to resveratrol being given as a dose of 20 mg/kg per day over 3 weeks. Also, DOX-related germ cell degeneration may be attributed to a low intratesticular testosterone concentration (Sharpe et al., 1992; Das et al., 2012) and administration of RES can ameliorate this damage. Histopathologically, this study demonstrated that 10 mg/kg−1 DOX led to disorganization in the germinal epithelium and the basal membrane of the seminiferous tubule, degeneration and vacuolization, and to loss of germ cells. It also caused a decrease in seminiferous tubule diameter, germinal epithelium diameter and JTBS. These findings support those of previous studies (Atessahin et al., 2006; Ceribasi et al., 2012). This histopathological damage may have caused disruption of spermatogenesis (Trivedi et al., 2011). Additionally, this situation might also be due to a decrease in sperm in semen (Gullen Unal et al., 2013). Apoptosis plays an important role in toxicity induced with DOX in various tissues.
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ROS leads to mitochondrial damage either directly, or else indirectly by affecting pro-apoptotic Bcl-2 proteins (Xin et al., 2012). In our study, AI rose significantly in the DOX group compared to the control group, and apoptosis was most pronounced in the spermatogonia together with other spermatogenic cells. Matsui et al. administered DOX treatment and reported apoptosis in all spermatogenic cells, particularly in type A spermatogonia (Matsui et al., 1993; Prahalathan et al., 2005a). The apoptotic index rising with DOX treatment may be attributed to increasing ROS and lipid peroxidation levels in testicular tissue. RES is a natural phytoalexin found in various plants. Studies have reported that it behaves as a ROS scavenger, metal chelator and enzyme regulator (Venturini et al., 2010). RES may enhance the activities of various antioxidant enzymes (Tunali-Akbay et al., 2010). Antioxidant enzymes such as SOD and CAT react against radicals such as superoxide and hydrogen peroxide, while GP-x has a scavenging effect against alkyl, alkoxy and peroxyl radicals that can form from oxidized membrane compounds and uses GSH as a substrate (Ceribasi et al., 2012). RES’s antioxidant activity may be ascribed to its ability to inhibit reductase in the ribonucleotide and cyclooxygenase 2 transcription ability in DNA polymerase activity (Uguralp et al., 2005a). One study involving diabetic rats reported that regular daily administration of resveratrol reduced lipid peroxidation and exhibited antioxidant activities in the rat hippocampus and frontal cortex (Venturini et al., 2010). A study investigating cardiotoxicity of cisplatin (CP) in rats reported that RES reduced myocardial injury, and prevented an increase in lactate dehydrogenase, a decrease in antioxidant enzyme activities and a rise in MDA. Additionally, RES was shown to have a protective effect against cytotoxicity induced with CP in cancer cells (Wang et al., 2009). In our study, plasma and tissue MDA levels were significantly lower in the DOX + RES group, while GP-x activity increased. Previous studies have reported that RES lowers MDA levels and reduces oxidative stress by increasing GSH levels (Uguralp et al., 2005a). One study also reported that RES may function like the antioxidant enzymes SOD1 and GPx1 (Spanier et al., 2009). These findings show that RES administration reduces lipid peroxidation induced by DOX. The increase in GP-x activity in testicular tissue observed in this study may also be explained by excessive production of this antioxidant in order to scavenge the overproduction of free radicals caused by oxidative stress (Ceribasi et al., 2012). Juan et al. suggested that transresveratrol has a therapeutic effect on spermatogenesis and the testis in adult rats. They also reported an increase in the production of spermatozoa with oral RES treatment in rats (Juan et al., 2005). Another study reported that RES exhibited a protective effect against lipid peroxidation and that RES can be added to new sperm culture media to protect sperm against ROS attacks for mechanical techniques such as semen cryopreservation or in vitro fertilization-intracytoplasmic sperm injection (Collodel et al., 2011). One study investigating the effect on testicular damage of the industrial toxin 2,5-hexanedione (2,5-HD) suggested that RES can alleviate 2,5-HD-induced dyszoospermia and regulate c-kit, a specific cell membrane protein (Jiang et al., 2008). In parallel with the literature, in our study observed an increase in sperm concentration and motility and a decrease in abnormal sperm count in the DOX + RES group compared to the DOX group. RES has also been shown to protect sperm against apoptosis resulting from physiological injury and to increase sperm production (Jiang et al., 2008). The antiapoptotic effect of RES has been shown in various experimental models (Uguralp et al., 2005b; Bitgul et al., 2013). Yulug et al. (2013) reported that RES can prevent the formation of ROS, and thus causes a decrease in TAI. Additionally, Uguralp et al. (2005a) suggested that RES reduced germ cell apoptosis in the testis after testicular ischemia/reperfusion injury. In our study, the
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administration of RES significantly reduced cell apoptosis. These effects are associated with its reduction of DNA breaks due its function as a scavenger of hydroxyl radicals (Uguralp et al., 2005b). Histologically, our study showed that RES treatment prevented DOX-related testicular damage. Testicular morphology and testis weight in the DOX + RES group were similar to those in the control group. The protective effect of RES treatment may be due to its protection of cellular membranes against oxidative damage (Yulug et al., 2013). In conclusion, this study shows that DOX treatment compromises normal testicular function, while RES makes a positive contribution to fertility by exhibiting anti-apoptotic and antiperoxidative effects against this damage. Financial disclosure The authors declare that they have no relevant financial interests. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgement This study was supported by the Scientific Researches Fund of Karadeniz Technical University (no. 2010.114.001.12). References Aebi DJ. Glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, editor. Methods of enzymatic analysis. 2nd ed. Weinheim: VCH Verlagsgesellschaft mbH; 1986a. p. 190–6. Aebi GU. Sorbitol dehydrogenase. In: Bergmeyer HU, editor. Methods of enzymatic analysis. 2nd ed. Weinheim: VCH Verlagsgesellschaft mbH; 1986b. p. 112–7. Aebi H. Catalase. In: Bergmeyer HU, editor. Methods of enzymatic analysis. New York: Academic Press; 1974. p. 673–7. Afzal M, Afzal A, Jones A, Armstrong D. A rapid method for the quantification of GSH and GSSG in biological samples. Methods Mol Biol 2002;186:117–22. Atessahin A, Türk G, Karahan I, Yılmaz S, Ceribas¸ı AO, Bulmus O. Lycopene prevents adriamycin-induced testicular toxicity in rats. Fertil Steril 2006;1:1216–22. Au WW, Hsu TC. The genotoxic effects of adriamycin in somatic and germinal cells of the mouse. Mutat Res 1980;79:351–61. Ayla S, Seckin I, Tanriverdi G, Cengiz M, Eser M, Soner BC, et al. Doxorubicin induced nephrotoxicity: protective effect of nicotinamide. Int J Cell Biol 2011;2011:390238. Benayahoum A, Amira-Guebailia H, Houache O. A DFT method for the study of the antioxidant action mechanism of resveratrol derivatives. J Mol Model 2013;19:2285–98. Belguendouz L, Fremont L, Gozzelıno MT. Interaction of trans resveratrol with plasma proteins. Biochem Pharmacol 1998;55:811–6. Bitgul G, Tekmen I, Keles D, Oktay G. Protective effects of resveratrol against chronic immobilization stress on testis. ISRN Urol 2013:278720. Caruso F, Tanski J, Villegas-Estrada A, Rossi M. Structural basis for antioxidant activity of trans-resveratrol: ab initio calculations and crystal and molecular structure. J Agric Food Chem 2004;52:7279–85. Ceribasi AO, Sakin F, Türk G, Sönmez M, Atessahin A. Impact of ellagic acid on adriamycin-induced testicular histopathological lesions, apoptosis, lipid peroxidation and sperm damages. Exp Toxicol Pathol 2012;64:717–24. Collodel G, Federicoa MG, Geminiania M, Martinic S, Bonechic C, Rossic C, et al. Effect of trans-resveratrol on induced oxidative stress in human sperm and in rat germinal cells. Reprod Toxicol 2011;31:239–46. Das J, Ghosh J, Manna P, Parames CS. Taurine protects rat testes against doxorubicininduced oxidative stress as well as p53, Fas and caspase 12-mediated apoptosis. Amino Acids 2012;42:1839–55. Doroshow JH, Locker GY, Myers CE. Enzymatic defenses of the mouse heart against reactive oxygen metabolites. Alterations produced by doxorubıcın. J Clin Invest 1980:128–35. Dudka J, Gieroba R, Korga A, Burdan F, Matysiak W, Jodlowska-Jedrych B, et al. Different effects of resveratrol on dose-related doxorubicin-induced heart and liver toxicity. Evid Based Complement Alternat Med 2012:606183. Frémont L. Biological effects of resveratrol. Life Sci 2000;66:663–73. Gharanei M, Hussain A, Janneh O, Maddock HL. Doxorubicin induced myocardial injury is exacerbated following ischaemic stress via opening of the mitochondrial permeability transition pore. Toxicol Appl Pharmacol 2013:149–56.
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Effects of resveratrol on doxorubicin induced testicular damage in rats夽 Sibel Türedi a,∗ , Esin Yulu˘g a , Ahmet Alver b , Ömer Kutlu c , Cemil Kahraman b a b c
Karadeniz Technical University, Faculty of Medicine, Department of Histology and Embryology, 61080 Trabzon, Turkey Karadeniz Technical University, Faculty of Medicine, Department of Medical Biochemistry, 61080 Trabzon, Turkey Karadeniz Technical University, Faculty of Medicine, Department of Urology, 61080 Trabzon, Turkey
a r t i c l e
i n f o
Article history: Received 12 March 2014 Accepted 8 December 2014 Keywords: Doxorubicin Testicular toxicity Apoptosis Resveratrol Infertility
a b s t r a c t The purpose of this study was to evaluate the likely protective effect of resveratrol (RES) on doxorubicin (DOX) induced testicular damage. Rats were divided into five groups: control, RES, dimethyl sulfoxide (DMSO), DOX and DOX + RES. At the end of treatment, the rats were sacrificed. Plasma testosterone levels, oxidative status, epididymal sperm parameters and testicular apoptosis were evaluated. MDA levels, GPx and GSH activities were higher in the DOX group than in the control group. MDA levels were lower in the DOX + RES group than in the DOX group. The DOX group exhibited a significant decrease in plasma testosterone levels, sperm concentration and motility, and a significant increase in abnormal sperm rate and TUNEL (+) cells in the testis. A significant increase was observed in plasma testosterone levels and sperm concentration and motility, and a significant decrease in the abnormal sperm rate and TUNEL (+) cells in the DOX + RES group compared to the DOX group. A marked improvement in severe degenerative alterations in the germinative epithelium was also observed following treatment with RES. In conclusion, RES makes a positive contribution to fertility by exhibiting anti-apoptotic and antiperoxidative effects against DOX-induced testicular damage. © 2014 Published by Elsevier GmbH.
1. Introduction Anticancer therapy generally compromises physiological hemostasis and causes multi-organ failure during the therapeutic process (Ayla et al., 2011). Doxorubicin (DOX) is an antracyclinegroup antibiotic that has been used as an antineoplastic agent in the treatment of various hematological malignities, including solid tumors, for many years (Doroshow et al., 1980; Gharanei et al., 2013). Due to its severe side-effects on the testis, as well as toxicity in various organs (Yeh et al., 2009), its clinical use is limited, however (Miranda et al., 2003; Ayla et al., 2011). DOX treatment may damage male fertility by adversely affecting sperm development, production and count (Prahalathan et al., 2005a). Previous studies have shown that it causes apoptosis in the seminiferous epithelium cycle (Sjoblom et al., 1998) and chromosomal damage in germ cells (Au and Hsu, 1980). The exact mechanism responsible
夽 The abstract of this study was presented at the 2012 XIth National Histology & Embryology Congress in Turkey (with international contribution). ∗ Corresponding author. Tel.: +90 462 3777736; fax: +90 462 3252270. E-mail addresses: [email protected], [email protected] (S. Türedi), [email protected] (E. Yulu˘g), [email protected] (A. Alver), [email protected] (Ö. Kutlu), [email protected] (C. Kahraman).
for DOX’s testicular toxicity is unclear, although recent findings point to oxidative stress and cellular apoptosis as major causes (Prahalathan et al., 2004; Yeh et al., 2009). DOX therapy-related oxidative damage is mediated by an oxyradical complex containing superoxide, hydroxyl radical and a certain amount of iron (Hida et al., 1995). Oxyradicals lead to damage in the mitochondrial and other cytoplasmic organelle membranes through proteins, nucleotides and phospholipids (Muraoka and Miura, 2003). Resveratrol (RES) (trans-3,4 ,5-trihidroksi-stilben) is a natural phytoalexin found in various plants, such as grape, peanut and mulberry (Krasnow and Murphy, 2004; Caruso et al., 2004). RES’ biological activities are known to exhibit antioxidant, antiinflammatory, anticancer, antiatherogenic and cardioprotective properties (Ignatowicz and Baer-Dubowska, 2001; Dudka et al., 2012; Benayahoum et al., 2013). In addition, RES’ antioxidant effect may also help protect against DNA damage occurring after a rise in ROS levels and against lipid peroxidation in the cell membrane (Jiang et al., 2008). Studies have shown that it is more effective that vitamins E and C, the best known antioxidants (Belguendouz et al., 1998). In another study, grapeseed extracts containing RES were shown to protect glial cells against oxidative stress (Royshowdhury et al., 2001). The antioxidant activity of RES is associated with its ability to inhibit ribonucleotide reductase and cyclooxygenase transcription in DNA polymerase activity (Frémont, 2000).
http://dx.doi.org/10.1016/j.etp.2014.12.002 0940-2993/© 2014 Published by Elsevier GmbH.
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The purpose of this study was to evaluate the likely protective effect of RES on DOX-induced testicular damage using histopathological and biochemical analyses. 2. Materials and methods 2.1. Animals and experimental design Thirty male Sprague–Dawley rats (5 weeks old, weighing 140–170 g) were used. All animals were kept in standard laboratory conditions. Standard laboratory chow and water were provided ad libitum throughout the experiment. The study was approved by the Institutional Animal Ethical Committee of Karadeniz Technical University, Trabzon, Turkey. The rats were randomly divided into five groups. The control group (n: 6) was given a single dose of 9% isotonic sodium chloride intraperitoneally (i.p.). The RES group (n: 6) was given 20 mg/kg RES i.p. (R5010-500 mg, Sigma–Aldrich, St. Louis, MO, USA) per day [dissolved in 30% dimethyl sulfoxide (DMSO)] every day throughout the experiment, while the DMSO group (n: 6) was administered 30% DMSO solution i.p. The DOX group (n: 6) was injected with a single dose of 10 mg/kg DOX (Adriblastina 10 mg, DEVA, Turkey) i.p. The DOX + RES group (n:6) was administered a single dose of 10 mg/kg DOX together with 20 mg/kg RES i.p. per day throughout the experiment. Dosages of RES (Oktem et al., 2012) and DOX (Atessahin et al., 2006) were determined on the basis of reports from previous studies. 2.2. Sample collection At the end of the 21-day experiment, rats were sacrificed by decapitation under ketamine (Ketalar, Pfizer, Turkey) anesthesia. The abdominal cavity was quickly opened and the testes extracted and separated from the epididymises. The right testicular epididymis was placed in a Petri dish containing 2 mL Tris buffer solution for sperm parameter analysis. The right testis and the left epididymis were fixed in Bouin’s solution for histological analysis. Blood and tissue specimens collected for biochemical parameters were stored at −80 ◦ C. 2.3. Biochemical analysis Plasma malondialdehyde (MDA) levels were determined using the method described by Yagi (1994). Tetramethoxypropane was used as a standard, and MDA levels were calculated as nmol/mL. Tissue MDA levels in testis samples were measured using the method described by Uchiyama and Mihara (1978). Tetramethoxypropane was used as a standard, and MDA levels were calculated as nanomoles per gram wet tissue. Superoxide dismutase (SOD) and catalase (CAT) activities were determined in the remaining part of the testis tissue. SOD activities were measured using the method described by Sun et al. (1988). Enzyme activity leading to 50% inhibition was taken as one unit, and the results were expressed as U/mg protein. CAT activity was determined using the method described by Aebi (1974), and the results were expressed as k/g protein (k, rate constant). Protein concentrations were determined following Lowrey’s method (1951). Glutathione peroxidase (GP-x) activity was determined using a commercial spectrophotometric kit (Cayman, USA). Glutathione (GSH) levels were analyzed using high-performance liquid chromatography (Afzal et al., 2002) (Agilent 1100 series HPLC and fluorescence detector systems, Waldbronn, Germany; column, Fortis UniverSil C18, 4.6 mm × 250 mm). Results were expressed as g/mL. Tissue glucose-6-phosphate dehydrogenase (G6PD) and sorbitol dehydrogenase (SDH) activities were measured using the methods described by Deutsch (Aebi, 1986a) and Gerlach (Aebi,
1986b), respectively. The results were expressed as U/g protein. Plasma testosterone levels were measured using a rat testosterone EIA kit (Cayman, USA).
2.4. Histopathological examinations Testis and epididymis specimens were fixed, embedded in paraffin blocks, sliced into 5-m sections and stained with hematoxylin–eosin (H&E) for histopathological evaluation. All testicular histology was examined by a histologist under light microscopy (Olympus BX-51; Olympus, Tokyo, Japan). For seminiferous tubule diameter (STD) and germinal epithelium thickness (GET) measurement, 10 different areas were selected at random from each testis section. The Analysis 5 Research program (Olympus Soft Imaging Solutions, Germany) was used under light microscopy at a magnification of 200×, and 20 seminiferous tubules were selected and measured on each testis section. Johnsen’s tubular biopsy score (JTBS) was used to evaluate spermatogenesis. Under this system, testis seminiferous tubule sections were scored from 0 to 10 (Johnsen, 1970).
2.5. TUNEL assay Testicular apoptosis was analyzed using the terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate nick end labeling assay (TUNEL) method. Sections 5 m in thickness were taken from the paraffin blocks and subjected to standard deparaffinization. TUNEL staining of sections was performed using an in situ cell death detection kit AP kit (Roche, Mannheim, Germany), in accordance with the manufacturer’s instructions. The proportion of TUNEL (+) spermatogenetic cells to the number of normal spermatogenetic cells was taken as the testis apoptotic index (TAI) (Karaguzel et al., 2012).
2.6. Epididymal sperm concentration, motility and abnormal sperm rate The epididymis was broken down in a Petri dish containing 2 mL Tris buffer tampon solution and incubated for 30 min at 37 ◦ C for the sperm to swim in the fluid. Counts, motility and abnormal sperm levels were calculated. A Makler sperm counting chamber (Sefi-Medical Instrument, Haifa, Israel) was used to calculate sperm concentrations. Briefly, 5 L of semen from the homogenate was dropped into the center of the chamber and the glass lid was closed. Sperm were made to swim at a depth of 10 m with the assistance of four quartz blades. Sperm count was then calculated under a light microscope at 200× magnification (%106 ). Sperm motility was calculated by modifying the method described by Sonmez et al. (2005). One drop of sperm fluid diluted in Tris buffer solution was collected by pipette, placed onto a slide and covered with a slide cover. Sperms were evaluated as motile or immotile under a light microscope at 400× magnification. Three different areas were selected for each specimen, and the mean of these was taken as the motility score. Sperm morphology was calculated by modifying the method described by Trivedi et al. (2011). Sperm containing solution was centrifuged at 1000 rpm for 3 min. Subsequently, 2–3 drops of solution were spread on a slide, dried, fixed with methanol for 3 min and stained with 1% eosin-y. Two hundred sperm from each animal were examined for head and tail anomalies under a light microscope under immersion oil at a magnification of 100×, and were classified as normal, head abnormal or tail abnormal (Xin et al., 2012).
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Table 1 Clinical signs, seminiferous tubule diameter, germinal epithelium thickness, testis histopathologic damage scores and apoptotic index in all groups. Parameters
Control (n:6)
Body weight (g) Testis weight (g) STD GET JTBS TAI (%)
212.8 1.43 259 90 9.73 2.74
± ± ± ± ± ±
RES (n:6)
16.76 0.17 4.37 4.47 0.37 1.13
189.5 1.25 238.3 87.5 9.76 3.11
± ± ± ± ± ±
DMSO (n:6)
12.53 0.08 4.72 2.42 0.15 1.02
189.6 1.25 241 86.57 9.63 3.04
± ± ± ± ± ±
DOX (n:6)
15.31 0.1 9.1 3.34 0.18 0.87
150.1 0.61 151.4 49.02 5.68 56.16
± ± ± ± ± ±
DOX + RES (n:6)
16.72a 0.13a 14.6a 6.80a 0.62a 5.11a
159.2 1.04 224 82.87 8.97 20.44
± ± ± ± ± ±
13.96a 0.14a , b 4.72b 2.57b 0.48b 2.65a , b
The data represent the mean ± SD. a P < 0.05 compared with control. b P < 0.05 compared with DOX group.
Table 2 Oxidant–antioxidant biochemical parameteres and plasma testosterone levels in all groups. Parameters Plasma testosterone (Pg/mL) Plasma MDA (nmol/L) Tissue MDA (nmol/g) GP-x (U/mg protein) GSH (g/mL) SOD (U/g tissue) CAT (k/g)
Control (n:6) 1025 0.68 377.4 1.43 4.19 6.66 0.02
± ± ± ± ± ± ±
288.5 0.19 44.35 0.16 0.07 1.77 0.03
RES (n:6) 821.6 0.74 385.7 1.39 4.41 5.96 0.01
± ± ± ± ± ± ±
36.3 0.44 42.61 0.42 0.33 1.41 0.002
DMSO (n:6) 848.9 0.62 347 1.67 4.18 5.80 0.01
± ± ± ± ± ± ±
99.7 0.19 62.58 0.49 0.19 0.82 0.002
DOX (n:6) a
428.5 1.30a 503a 2.01a 5.86a 5.07 0.007
± ± ± ± ± ± ±
129.8 0.48 27.45 0.34 0.33 1.82 0.001
DOX + RES (n:6) 800.5b 0.85b 448.3b 2.19a , b 6.31a 5.26 0.10
± ± ± ± ± ± ±
261.6 0.10 48.23 0.26 0.71 1.56 0.002
The data represent the mean ± SD. a P < 0.05 compared with control. b P < 0.05 compared with DOX group.
2.7. Statistical analysis All statistical analyses were performed using computer software (Prism 5.0, Graphpad Software Inc., San Diego, CA, USA). Data were presented as mean (±) standard deviation (SD). Kruskal–Wallis analysis of variance and the Mann–Whitney U-test with Bonferroni correction were used for statistical analysis. Significance was set at P < 0.05.
3. Results 3.1. Clinical signs Body weight in the DOX group decreased significantly compared to the control group (P < 0.05). Although an increase was observed in the DOX + RES group compared to the DOX group, this was not significant (Table 1). Testis weight in the DOX group decreased significantly compared to the control group. DOX + RES group values were significantly higher compared to those of the DOX group (P < 0.05) (Table 1).
3.2. Biochemical analysis results Experimental group plasma testosterone, plasma MDA and tissue MDA levels and SOD, CAT, GP-x and GSH activity findings are shown in Table 2. Plasma testosterone levels decreased significantly in the DOX group compared to the control group. A significant increase was observed in the DOX + RES group compared to the DOX group (P < 0.05). Plasma and tissue MDA levels and GP-x and GSH activities increased significantly in the DOX group compared to the control group. Plasma and tissue MDA levels decreased significantly in the DOX + RES group compared to the DOX group (P < 0.05), while GP-x activity rose significantly. No significant difference was determined in testicular tissue SOD and CAT activities between the groups (P > 0.05). In addition, although a decrease was observed in G6PD and SDH enzyme levels in the DOX group (0.09 ± 0.66 and 2.65 ± 1.19, respectively)
compared to the control group (0.19 ± 0.05 and3.49 ± 2.05, respectively), this was not significant. 3.3. Histopathological findings Testis and epididymis tissues in the control (Fig. 1A and D), RES and DMSO groups had a normal histological appearance. Disorganization in the seminiferous tubule basal membrane and seminiferous epithelium, vacuolization, a decrease in germinal cells, and unmatured germinal epithelial cells in the lumen were observed in the DOX group (Fig. 1B). Few spermatozoa were seen in the lumen in the epididymal tissue of the DOX group (Fig. 1E). In the DOX + RES group, although there were a few tubules in which non-matured germinal epithelial cells were observed in the lumen (Fig. 1C and F), the appearance was close to that of the control group. In the DOX group, STD and GET decreased significantly compared to the control group (P < 0.05). A significant increase was observed in the DOX + RES group compared with the DOX group (P < 0.05) (Table 1). JTBS in the DOX group decreased significantly compared to the control group (P < 0.05). A significant increase was observed in the DOX + RES group compared to the DOX group (P < 0.05) (Table 1). TAI rose significantly in the DOX group compared to the control group. A significant decrease was seen in the DOX + RES group compared to the DOX group (P < 0.05) (Table 1). TUNEL (+) cells in the DOX group were widespread in the germinal epithelial cells of the seminiferous tubule (Fig. 2B). There was a significant decrease in TUNEL (+) cells in the DOX + RES group (Fig. 2C). 3.4. Epididymal sperm concentration, motility and abnormal sperm rate Sperm concentration, motility and abnormal sperm rate results are shown in Table 3. Following DOX administration, a significant decrease in sperm concentration and motility and a significant increase in abnormal sperm numbers were observed in comparison to the control group (P < 0.05). A significant increase in sperm concentration and motility, and a significant decrease in abnormal
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Fig. 1. Testis and epididymal section micrographs: (A, D: control group; B, E: DOX group; C, F: DOX + RES group): (A) normal seminiferous tubule germinal epithelium (arrow) and spermatozoa in the lumen (arrowhead); (B) vacuoliation and degeneration in the germinal epithelium (arrow), decreased spermatozoa (arrowhead) and germinal cell clustering in the lumen (stars); (C) nearly normal seminiferous tubule germinal epithelium (arrow), very few immature germinal cells (star) and spermatozoa (arrowhead) in the lumen (H&E 200×); (D) normal epididymal histology; (E) decreased spermatozoa (stars) and spermatids clustering in the epididymal lumen (arrows); (F) spermatozoa (arrowhead) and very few spermatids (arrows) in the epididymal lumen (H&E 400×).
Fig. 2. Testis seminiferous tubule sections stained with the TUNEL technique (original magnification 400×) (A: control group; B: DOX group; C: DOX + RES group). Apoptotic spermatogenetic cells (arrows).
Table 3 Epididymal sperm concentration, motility, abnormal sperm rate in all groups. Parameters
Control (n:6)
RES (n:6)
DMSO (n:6)
Sperm concentration (×106 ) Motility (%) Abnormal sperm rate (%)
22.83 ± 6.52 73.1 ± 9.23 2.48 ± 0.09
20.17 ± 0.75 68.34 ± 9.86 2.31 ± 0.20
19.67 ± 1.5 71.53 ± 8.99 2.21 ± 0.14
DOX (n:6) 4.5a ± 3.50 8.10a ± 15.07 5.80a ± 0.85
DOX + RES (n:6) 15.14a , b ± 3.97 57.86b ± 6.46 3.89b ± 0.83
The data represent the mean ± SD. a P < 0.05 compared with control. b P < 0.05 compared with DOX group.
sperm count were observed in the DOX + RES group compared to the DOX group (P < 0.05).
4. Discussion Testicular dysfunction is one of the serious side-effects of cytotoxic chemotherapy (Prahalathan et al., 2005b). DOX is a widely used cytotoxic agent known to impair testicular function and spermatogenesis (Ceribasi et al., 2012). Protection of fertility is important during the use of chemotherapeutics. The present study is an attempt to evaluate the role of resveratrol in DOX-induced testicular dysfunction in rats. In our study, testis weight decreased significantly in the DOX administered group. Kato et al. (2001) observed a decreased in testis weight in chronic exposure to DOX. We think that this decrease after DOX administration may be due to severe parenchymal atrophy in the testicular seminiferous tubule (Sudha and Kavimani, 2011).
ROS species such as superoxide ions, hydroxyl radical, peroxyl and alkoxy radicals and the non-radical oxygen, hydrogen peroxide and ozone are produced during oxygen use in normal metabolism, and are required for various physiological conditions in the male reproductive system. However, excessive production of ROS leads to lipid peroxidation, and this results in oxidative stress. Cells employ various antioxidant mechanisms to reduce the levels of excess ROS. Antioxidant enzymes such as SOD and CAT react against radicals such as superoxide and hydrogen peroxide, while GP-x has a scavenging effect against alkyl, alkoxy and peroxyl radicals that can form from oxidized membrane compounds, and uses GSH as a substrate (Ceribasi et al., 2012). Previous studies have reported that oxidative stress is the mechanism responsible for toxicity developing in testicular tissue due to DOX (Lebrecht et al., 2007; Trivedi et al., 2011). In present study, a significant increase in MDA levels was observed in the DOX group, indicating increased lipid peroxidation (Ceribasi et al., 2012). This suggests that the increase may be due to DOX-associated excessive ROS production and a rise in lipid
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peroxidation. GSH plays an important role in the detoxification of free radicals and of ROS’ antioxidative and xenobiotic compounds (Ayla et al., 2011). Salvemini et al. (1999) reported that GSH synthesis may develop as an adaptation process in situations in which cells are exposed to oxidative stress. Similarly, Yilmaz et al. (2006) confirmed that GSH synthesis may be a positive regulation under oxidative stress resulting in a rise in GSH concentrations. GP-x and GSH activities also rose significantly in the DOX group compared to the control group in our study. Although a decrease was seen in SOD and CAT activities, this was not statistically significant. The increase in GP-x and GSH activities in this study may be attributed to excess production in order to scavenge free radicals produced in excess quantities under oxidative stress (Ceribasi et al., 2012). SDH is involved in the provision of energy for sperm cells and converts sorbitol and fructose. A decrease in the activity of this enzyme after DOX treatment compromises the distribution of energy metabolism in sperm cells. Another key enzyme in testicular tissue is G6PD, which gives rise to an equivalent decrease for the hydroxylation of steroids. G6PD is directly involved in glutathione metabolism (Das et al., 2012). Low levels of this enzyme increase oxidative stress and may lead to cell death. Prahalathan et al. (2004) reported lower levels of this enzyme in a group treated with DOX compared to a control group. In our study, while G6PD and SDH enzymes were lower in the DOX group compared to the control group, the difference was not significant. The changes observed after DOX administration in a previous study indicated serious oligospermia in the epididymis after 4 weeks and azoospermia after 9 weeks (Zanetti et al., 2007). In this study, sperm concentration and motility decreased significantly and the abnormal sperm rate rose significantly in the DOX group. Another previous study demonstrated that 10 mg/kg−1 DOX treatment led to a decreased in motility, but not in sperm concentration (Atessahin et al., 2006). Spermatozoa plasma membranes contain large amounts of saturated fatty acids, and antioxidant enzyme levels in their cytoplasm are very low. Oxidative stress, which exhibits high sensitivity for denaturation and fragmentation, may play a significant role in the emergence of sperm anomalies by affecting sperm DNA (Sikka, 1996). Previous studies have shown that DOX causes DNA fragmentation and chromosome damage (Menna et al., 2010; Xin et al., 2012) and that increasing oxidative stress causes a decrease in the number of motile sperm and a rise in abnormal sperm rates (Atessahin et al., 2006). Testosterone is needed for spermatogenesis, the establishment of structural morphology and the physiology of the seminiferous tubule (Das et al., 2012). Juan et al. (2005) observed an increase in serum testosterone and gonadotropin levels, a decrease in tubular density and a significant increase in sperm numbers of rats given resveratrol at 20 mg/kg per day over 90 days. In our study, there was a significant increase in sperm maturation and plasma testosterone levels in rats given DOX and concomitant resveratrol compared to the DOX group. This may be due to resveratrol being given as a dose of 20 mg/kg per day over 3 weeks. Also, DOX-related germ cell degeneration may be attributed to a low intratesticular testosterone concentration (Sharpe et al., 1992; Das et al., 2012) and administration of RES can ameliorate this damage. Histopathologically, this study demonstrated that 10 mg/kg−1 DOX led to disorganization in the germinal epithelium and the basal membrane of the seminiferous tubule, degeneration and vacuolization, and to loss of germ cells. It also caused a decrease in seminiferous tubule diameter, germinal epithelium diameter and JTBS. These findings support those of previous studies (Atessahin et al., 2006; Ceribasi et al., 2012). This histopathological damage may have caused disruption of spermatogenesis (Trivedi et al., 2011). Additionally, this situation might also be due to a decrease in sperm in semen (Gullen Unal et al., 2013). Apoptosis plays an important role in toxicity induced with DOX in various tissues.
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ROS leads to mitochondrial damage either directly, or else indirectly by affecting pro-apoptotic Bcl-2 proteins (Xin et al., 2012). In our study, AI rose significantly in the DOX group compared to the control group, and apoptosis was most pronounced in the spermatogonia together with other spermatogenic cells. Matsui et al. administered DOX treatment and reported apoptosis in all spermatogenic cells, particularly in type A spermatogonia (Matsui et al., 1993; Prahalathan et al., 2005a). The apoptotic index rising with DOX treatment may be attributed to increasing ROS and lipid peroxidation levels in testicular tissue. RES is a natural phytoalexin found in various plants. Studies have reported that it behaves as a ROS scavenger, metal chelator and enzyme regulator (Venturini et al., 2010). RES may enhance the activities of various antioxidant enzymes (Tunali-Akbay et al., 2010). Antioxidant enzymes such as SOD and CAT react against radicals such as superoxide and hydrogen peroxide, while GP-x has a scavenging effect against alkyl, alkoxy and peroxyl radicals that can form from oxidized membrane compounds and uses GSH as a substrate (Ceribasi et al., 2012). RES’s antioxidant activity may be ascribed to its ability to inhibit reductase in the ribonucleotide and cyclooxygenase 2 transcription ability in DNA polymerase activity (Uguralp et al., 2005a). One study involving diabetic rats reported that regular daily administration of resveratrol reduced lipid peroxidation and exhibited antioxidant activities in the rat hippocampus and frontal cortex (Venturini et al., 2010). A study investigating cardiotoxicity of cisplatin (CP) in rats reported that RES reduced myocardial injury, and prevented an increase in lactate dehydrogenase, a decrease in antioxidant enzyme activities and a rise in MDA. Additionally, RES was shown to have a protective effect against cytotoxicity induced with CP in cancer cells (Wang et al., 2009). In our study, plasma and tissue MDA levels were significantly lower in the DOX + RES group, while GP-x activity increased. Previous studies have reported that RES lowers MDA levels and reduces oxidative stress by increasing GSH levels (Uguralp et al., 2005a). One study also reported that RES may function like the antioxidant enzymes SOD1 and GPx1 (Spanier et al., 2009). These findings show that RES administration reduces lipid peroxidation induced by DOX. The increase in GP-x activity in testicular tissue observed in this study may also be explained by excessive production of this antioxidant in order to scavenge the overproduction of free radicals caused by oxidative stress (Ceribasi et al., 2012). Juan et al. suggested that transresveratrol has a therapeutic effect on spermatogenesis and the testis in adult rats. They also reported an increase in the production of spermatozoa with oral RES treatment in rats (Juan et al., 2005). Another study reported that RES exhibited a protective effect against lipid peroxidation and that RES can be added to new sperm culture media to protect sperm against ROS attacks for mechanical techniques such as semen cryopreservation or in vitro fertilization-intracytoplasmic sperm injection (Collodel et al., 2011). One study investigating the effect on testicular damage of the industrial toxin 2,5-hexanedione (2,5-HD) suggested that RES can alleviate 2,5-HD-induced dyszoospermia and regulate c-kit, a specific cell membrane protein (Jiang et al., 2008). In parallel with the literature, in our study observed an increase in sperm concentration and motility and a decrease in abnormal sperm count in the DOX + RES group compared to the DOX group. RES has also been shown to protect sperm against apoptosis resulting from physiological injury and to increase sperm production (Jiang et al., 2008). The antiapoptotic effect of RES has been shown in various experimental models (Uguralp et al., 2005b; Bitgul et al., 2013). Yulug et al. (2013) reported that RES can prevent the formation of ROS, and thus causes a decrease in TAI. Additionally, Uguralp et al. (2005a) suggested that RES reduced germ cell apoptosis in the testis after testicular ischemia/reperfusion injury. In our study, the
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