Experimental Chemotherapy Received: March 20, 2014 Accepted after revision: July 03, 2014 Published online: October 4, 2014
Chemotherapy 2014;60:7–12 DOI: 10.1159/000365727
Protective Effects of Rosmarinic Acid on Doxorubicin-Induced Testicular Damage Ummugul Uyeturk a Ugur Uyeturk b Tulin Firat c Ayhan Cetinkaya d Buket Kin Tekce e Serkan Cakir f
Departments of a Medical Oncology, b Urology, c Histology, d Laboratory Animal Science, e Biochemistry and f Laboratory Animal Science, Abant Izzet Baysal University, Bolu, Turkey
Abstract Background: We investigated the protective effects of rosmarinic acid (RA) on testicular damage induced by doxorubicin (DXR) in rats. Methods: In total, 21 rats were divided into 3 groups: the control group that received no treatment, the DXR group that received intraperitoneal (i.p.) DXR on day 7 and the DXR + RA group that received intragastric RA for 10 days with i.p. DXR on day 7. The rats were sacrificed on day 11 for histological and biochemical analyses. To assess oxidative damage, glutathione peroxidase (GPx) and malondialdehyde (MDA) levels were measured. Results: The median modified Johnsen score of the DXR + RA group was higher than that of the DXR group (p = 0.002). The rats with the narrowest seminiferous tubules were in the DXR group (0.17 ± 0.03), and the difference between the DXR + RA and DXR groups was statistically significant (p = 0.002). The number of apoptotic cells in the DXR group was significantly higher than that in the control group, and there were significantly fewer apoptotic cells in the DXR + RA group than in the DXR group (p = 0.002). The MDA level was lowest in the control group and highest in the DXR group, and the level observed in the DXR + RA group significantly lower than that in the DXR group (p = 0.002). The GPx level was highest in the control group, with the level
© 2014 S. Karger AG, Basel 0009–3157/14/0601–0007$39.50/0 E-Mail [email protected] www.karger.com/che
observed in the DXR + RA group significantly higher than that in the DXR group (p = 0.002). The testosterone level was lowest in the DXR group and highest in the control group, and that observed in the DXR + RA group was significantly higher than that in the DXR group (p = 0.018). Conclusions: RA can correct DXR-induced testicular damage through its antioxidant properties. However, the mechanism underlying the effects of RA requires further investigation, and long-term and comparative human studies are © 2014 S. Karger AG, Basel also needed.
Introduction
Doxorubicin (DXR), an antitumor drug of the anthracycline class, has been used as an antineoplastic agent for over 4 decades. This drug is widely used in solid tumors including breast, uterine, prostate and ovarian tumors as well as in osteosarcoma, soft-tissue sarcoma, Kaposi’s sarcoma and hematological malignancies such as leukemia and lymphoma [1]. DXR interferes with the enzymes involved in DNA replication regardless of the cell cycle phase, although it is most active against mitotic cells [2]. However, the effects of this agent are not specific to the tumor targeted for treatment, and toxic effects are commonly observed in the heart, brain, liver, kidneys and testes [3]. This drug also causes an increase in the numbers Associate Prof. Ummugul Uyeturk, MD Department of Medical Oncology, Faculty of Medicine Abant Izzet Baysal University TR–14280 Bolu (Turkey) E-Mail ummuguluyeturk @ yahoo.com.tr
Downloaded by: UCSF Library & CKM 169.230.243.252 - 12/13/2014 11:26:11 AM
Key Words Testicular damage · Rosmarinic acid · Doxorubicin · Rat
Materials and Methods This study was conducted at the Experimental Animals Application and Investigation Center of Abant Izzet Baysal University. Approval from the local ethics committee was obtained. Eightweek-old Wistar albino male rats were used for the investigation and housed at an ambient temperature of 22 ± 2 ° C, under a 12/12hour light/dark cycle and 40–70% humidity. The rats were provided with pelleted rat food and tap water. RA (96%, No. 536954, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and DXR (DOXO-TEVA, Med-Ilac, Maslak, Turkey) were diluted in saline to obtain solutions of 50 mg/4 ml and 10 mg/10 ml, respectively. All of the intragastric (i.g.) administrations were performed after a 12-hour fast, and the animals were allowed free access to food 2.5 h after the procedure. The rats were randomized into 1 of 3 groups with 7 rats per group. • Group 1 (control) received 4 ml/kg/day i.g. saline for 10 days and 10 ml/kg/day intraperitoneal (i.p.) saline on day 7. • Group 2 (DXR) received 4 ml/kg/day i.g. saline for 10 days and 10 ml/kg/day i.p. DXR on day 7. • Group 3 (DXR + RA) received 50 mg/kg/day i.g. RA for 10 days and 10 ml/kg/day i.p. DXR on day 7.
8
Chemotherapy 2014;60:7–12 DOI: 10.1159/000365727
General appearance and mortality were observed daily, and body weights were recorded on days 1, 7 and 11. On day 11, all of the animals were euthanized by i.p. administration of 90 mg/kg ketamine and 10 mg/kg xylazine. The testes of the rats in all 3 groups were removed for histological and biochemical examinations, and blood was drawn by cardiac puncture for testosterone measurements. Histological Evaluation The right testis was taken and fixed in Bouin solution. Fixedtissue samples were dehydrated using a graded ethanol series, embedded in paraffin wax blocks, and sectioned at 3-mm thickness. The sections were then dewaxed with xylene, rehydrated using a graded ethanol series, and stained with hematoxylin and eosin. The germinal epithelia of at least 50 tubules were assessed according to a modified Johnsen score [11, 12]. Apoptosis in the spermatogenic cells of the seminiferous tubules was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nickend labeling (TUNEL). In each section, positively stained apoptotic cells in 10 seminiferous tubules were counted under a light microscope at ×20 magnification. The seminiferous tubular diameter (STD) was measured using a micrometer eyepiece, and the mean STD of randomly selected seminiferous tubules in each tissue section was calculated. All of the histopathological assessments were carried out by an experienced histologist who was blinded to the groups. Biochemical Evaluation Left testis samples were placed in glass tubes and labeled. After washing once with PBS, they were stored at –80 ° C until biochemical analysis. The samples were thawed before the assay, and 100 mg of testicular tissue was weighed. Each sample was homogenized in 1 ml of PBS. The tissues were frozen and thawed twice to rupture the cell membrane, and the homogenate was centrifuged at 5,000 g for 5 min at 2–8 ° C. The supernatant was removed and assayed for malondialdehyde (MDA) and glutathione peroxidase (GPx). MDA and GPx measurements were performed using commercial ELISA kits specific to rat tissues according to the manufacturer’s protocol (Cusabio, Wuhan, China). The linear ranges were 31.25–2,000 pmol/ml and 12.5–800 mIU/ml for MDA and GPx, respectively. For the testosterone analysis, blood samples were placed into separator tubes, incubated at room temperature for 30 min to ensure coagulation, and then centrifuged at 1,000 g for 15 min. Testosterone levels were measured in serum using commercial ELISA kits for endogenous rat testosterone according to the manufacturer’s protocol (Cusabio). All of the samples were assayed at the same time. The linear range was 0.13–25.6 ng/ml.
Statistical Analyses The data were analyzed using SPSS software v20 (Windows; SPSS Inc., Chicago, Ill., USA). The Shapiro-Wilk test was used to determine whether the distributions of continuous variables were normal. The Levene test was used to evaluate the homogeneity of variances. Data are shown as the means ± standard deviations or medians (interquartile range) where appropriate. The mean differences among groups were compared by one-way ANOVA or Welch ANOVA. Otherwise, the Kruskal-Wallis test was used for com-
Uyeturk/Uyeturk/Firat/Cetinkaya/ Kin Tekce/Cakir
Downloaded by: UCSF Library & CKM 169.230.243.252 - 12/13/2014 11:26:11 AM
of apoptotic cells and abnormal sperm, thus negatively impacting fertility [2]. Although the adverse effects of DXR are not fully understood, it is known to increase oxidative stress, which suggests that reactive oxygen species (ROS) likely play a role [4]. Oxidants from the environment (e.g. pollution and radiation), certain nutrients and various pathologies can generate ROS which, in addition to free radicals, can alter DNA, proteins and membrane phospholipids. Moreover, decreased levels of intracellular antioxidants during conditions of acute oxidative stress or in various disease states result in increased levels of intracellular ROS. It is thus important to balance the antioxidant-to-oxidant ratio by supplementation or induction of antioxidants to prevent the cellular damage associated with oxidative stress [5]. Rosmarinic acid (RA), which was purified 50 years ago from Rosmarinus officinalis, is a caffeic acid and 3,4-dihydroxyphenyllactic acid ester [6]. RA is commonly found in species of the Boraginaceae family and in the subfamily Nepetoideae of Lamiaceae, and it is also found in species of other higher plant families and in some fern and hornwort species [7, 8]. RA has a number of interesting biological properties, such as antiviral, antibacterial, antiinflammatory and antioxidant activities [7, 9]. Various studies have demonstrated that RA can alleviate damage in tissues exposed to toxic substances [8, 10], but none has explored the efficacy of RA on testicular damage secondary to DXR. Thus, we investigated the efficacy of RA in terms of preventing tissue injury induced by DXR.
Table 1. Comparison of the body weight and testicular weight of rats
Body weight Day 1 Day 7 Day 11 Testes weight
Control
DXR
DXR + RA
p value
174.6±9.5 249.1±10.3 270.1±9.6 2.6±0.14
177.1±10.4 249.9±9.9 226.7±8.7 2.0±0.15
178.3±5.4 247.0±9.3 257.9±8.3 2.3±0.11
0.50 0.94
Chemotherapy 2014;60:7–12 DOI: 10.1159/000365727
Protective Effects of Rosmarinic Acid on Doxorubicin-Induced Testicular Damage Ummugul Uyeturk a Ugur Uyeturk b Tulin Firat c Ayhan Cetinkaya d Buket Kin Tekce e Serkan Cakir f
Departments of a Medical Oncology, b Urology, c Histology, d Laboratory Animal Science, e Biochemistry and f Laboratory Animal Science, Abant Izzet Baysal University, Bolu, Turkey
Abstract Background: We investigated the protective effects of rosmarinic acid (RA) on testicular damage induced by doxorubicin (DXR) in rats. Methods: In total, 21 rats were divided into 3 groups: the control group that received no treatment, the DXR group that received intraperitoneal (i.p.) DXR on day 7 and the DXR + RA group that received intragastric RA for 10 days with i.p. DXR on day 7. The rats were sacrificed on day 11 for histological and biochemical analyses. To assess oxidative damage, glutathione peroxidase (GPx) and malondialdehyde (MDA) levels were measured. Results: The median modified Johnsen score of the DXR + RA group was higher than that of the DXR group (p = 0.002). The rats with the narrowest seminiferous tubules were in the DXR group (0.17 ± 0.03), and the difference between the DXR + RA and DXR groups was statistically significant (p = 0.002). The number of apoptotic cells in the DXR group was significantly higher than that in the control group, and there were significantly fewer apoptotic cells in the DXR + RA group than in the DXR group (p = 0.002). The MDA level was lowest in the control group and highest in the DXR group, and the level observed in the DXR + RA group significantly lower than that in the DXR group (p = 0.002). The GPx level was highest in the control group, with the level
© 2014 S. Karger AG, Basel 0009–3157/14/0601–0007$39.50/0 E-Mail [email protected] www.karger.com/che
observed in the DXR + RA group significantly higher than that in the DXR group (p = 0.002). The testosterone level was lowest in the DXR group and highest in the control group, and that observed in the DXR + RA group was significantly higher than that in the DXR group (p = 0.018). Conclusions: RA can correct DXR-induced testicular damage through its antioxidant properties. However, the mechanism underlying the effects of RA requires further investigation, and long-term and comparative human studies are © 2014 S. Karger AG, Basel also needed.
Introduction
Doxorubicin (DXR), an antitumor drug of the anthracycline class, has been used as an antineoplastic agent for over 4 decades. This drug is widely used in solid tumors including breast, uterine, prostate and ovarian tumors as well as in osteosarcoma, soft-tissue sarcoma, Kaposi’s sarcoma and hematological malignancies such as leukemia and lymphoma [1]. DXR interferes with the enzymes involved in DNA replication regardless of the cell cycle phase, although it is most active against mitotic cells [2]. However, the effects of this agent are not specific to the tumor targeted for treatment, and toxic effects are commonly observed in the heart, brain, liver, kidneys and testes [3]. This drug also causes an increase in the numbers Associate Prof. Ummugul Uyeturk, MD Department of Medical Oncology, Faculty of Medicine Abant Izzet Baysal University TR–14280 Bolu (Turkey) E-Mail ummuguluyeturk @ yahoo.com.tr
Downloaded by: UCSF Library & CKM 169.230.243.252 - 12/13/2014 11:26:11 AM
Key Words Testicular damage · Rosmarinic acid · Doxorubicin · Rat
Materials and Methods This study was conducted at the Experimental Animals Application and Investigation Center of Abant Izzet Baysal University. Approval from the local ethics committee was obtained. Eightweek-old Wistar albino male rats were used for the investigation and housed at an ambient temperature of 22 ± 2 ° C, under a 12/12hour light/dark cycle and 40–70% humidity. The rats were provided with pelleted rat food and tap water. RA (96%, No. 536954, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and DXR (DOXO-TEVA, Med-Ilac, Maslak, Turkey) were diluted in saline to obtain solutions of 50 mg/4 ml and 10 mg/10 ml, respectively. All of the intragastric (i.g.) administrations were performed after a 12-hour fast, and the animals were allowed free access to food 2.5 h after the procedure. The rats were randomized into 1 of 3 groups with 7 rats per group. • Group 1 (control) received 4 ml/kg/day i.g. saline for 10 days and 10 ml/kg/day intraperitoneal (i.p.) saline on day 7. • Group 2 (DXR) received 4 ml/kg/day i.g. saline for 10 days and 10 ml/kg/day i.p. DXR on day 7. • Group 3 (DXR + RA) received 50 mg/kg/day i.g. RA for 10 days and 10 ml/kg/day i.p. DXR on day 7.
8
Chemotherapy 2014;60:7–12 DOI: 10.1159/000365727
General appearance and mortality were observed daily, and body weights were recorded on days 1, 7 and 11. On day 11, all of the animals were euthanized by i.p. administration of 90 mg/kg ketamine and 10 mg/kg xylazine. The testes of the rats in all 3 groups were removed for histological and biochemical examinations, and blood was drawn by cardiac puncture for testosterone measurements. Histological Evaluation The right testis was taken and fixed in Bouin solution. Fixedtissue samples were dehydrated using a graded ethanol series, embedded in paraffin wax blocks, and sectioned at 3-mm thickness. The sections were then dewaxed with xylene, rehydrated using a graded ethanol series, and stained with hematoxylin and eosin. The germinal epithelia of at least 50 tubules were assessed according to a modified Johnsen score [11, 12]. Apoptosis in the spermatogenic cells of the seminiferous tubules was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nickend labeling (TUNEL). In each section, positively stained apoptotic cells in 10 seminiferous tubules were counted under a light microscope at ×20 magnification. The seminiferous tubular diameter (STD) was measured using a micrometer eyepiece, and the mean STD of randomly selected seminiferous tubules in each tissue section was calculated. All of the histopathological assessments were carried out by an experienced histologist who was blinded to the groups. Biochemical Evaluation Left testis samples were placed in glass tubes and labeled. After washing once with PBS, they were stored at –80 ° C until biochemical analysis. The samples were thawed before the assay, and 100 mg of testicular tissue was weighed. Each sample was homogenized in 1 ml of PBS. The tissues were frozen and thawed twice to rupture the cell membrane, and the homogenate was centrifuged at 5,000 g for 5 min at 2–8 ° C. The supernatant was removed and assayed for malondialdehyde (MDA) and glutathione peroxidase (GPx). MDA and GPx measurements were performed using commercial ELISA kits specific to rat tissues according to the manufacturer’s protocol (Cusabio, Wuhan, China). The linear ranges were 31.25–2,000 pmol/ml and 12.5–800 mIU/ml for MDA and GPx, respectively. For the testosterone analysis, blood samples were placed into separator tubes, incubated at room temperature for 30 min to ensure coagulation, and then centrifuged at 1,000 g for 15 min. Testosterone levels were measured in serum using commercial ELISA kits for endogenous rat testosterone according to the manufacturer’s protocol (Cusabio). All of the samples were assayed at the same time. The linear range was 0.13–25.6 ng/ml.
Statistical Analyses The data were analyzed using SPSS software v20 (Windows; SPSS Inc., Chicago, Ill., USA). The Shapiro-Wilk test was used to determine whether the distributions of continuous variables were normal. The Levene test was used to evaluate the homogeneity of variances. Data are shown as the means ± standard deviations or medians (interquartile range) where appropriate. The mean differences among groups were compared by one-way ANOVA or Welch ANOVA. Otherwise, the Kruskal-Wallis test was used for com-
Uyeturk/Uyeturk/Firat/Cetinkaya/ Kin Tekce/Cakir
Downloaded by: UCSF Library & CKM 169.230.243.252 - 12/13/2014 11:26:11 AM
of apoptotic cells and abnormal sperm, thus negatively impacting fertility [2]. Although the adverse effects of DXR are not fully understood, it is known to increase oxidative stress, which suggests that reactive oxygen species (ROS) likely play a role [4]. Oxidants from the environment (e.g. pollution and radiation), certain nutrients and various pathologies can generate ROS which, in addition to free radicals, can alter DNA, proteins and membrane phospholipids. Moreover, decreased levels of intracellular antioxidants during conditions of acute oxidative stress or in various disease states result in increased levels of intracellular ROS. It is thus important to balance the antioxidant-to-oxidant ratio by supplementation or induction of antioxidants to prevent the cellular damage associated with oxidative stress [5]. Rosmarinic acid (RA), which was purified 50 years ago from Rosmarinus officinalis, is a caffeic acid and 3,4-dihydroxyphenyllactic acid ester [6]. RA is commonly found in species of the Boraginaceae family and in the subfamily Nepetoideae of Lamiaceae, and it is also found in species of other higher plant families and in some fern and hornwort species [7, 8]. RA has a number of interesting biological properties, such as antiviral, antibacterial, antiinflammatory and antioxidant activities [7, 9]. Various studies have demonstrated that RA can alleviate damage in tissues exposed to toxic substances [8, 10], but none has explored the efficacy of RA on testicular damage secondary to DXR. Thus, we investigated the efficacy of RA in terms of preventing tissue injury induced by DXR.
Table 1. Comparison of the body weight and testicular weight of rats
Body weight Day 1 Day 7 Day 11 Testes weight
Control
DXR
DXR + RA
p value
174.6±9.5 249.1±10.3 270.1±9.6 2.6±0.14
177.1±10.4 249.9±9.9 226.7±8.7 2.0±0.15
178.3±5.4 247.0±9.3 257.9±8.3 2.3±0.11
0.50 0.94
