http://informahealthcare.com/aan ISSN: 1939-6368 (print), 1939-6376 (electronic) Syst Biol Reprod Med, Early Online: 1–6 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/19396368.2014.930212
RESEARCH COMMUNICATION
The effect of melatonin on procarbazine induced testicular toxicity on rats Bilal Fırat Alp1*, Vural Kesik2, Ercan Malkoc¸1, Nuri Yig˘it3, Mehmet Saldır4, Og˘uzhan Babacan2, Emin O¨zgu¨r Akgu¨l5, Yavuz Poyrazoglu6, Nadir Korkmazer4, Mustafa Gulgun4, and Onur Erdem7
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Department of Urology, 2Pediatric Hematology and Oncology, 3Pathology, 4Pediatrics, 5Biochemistry, 6Surgery, and 7Toxicology, Gulhane Military Medical Faculty, Ankara, Turkey Abstract
Keywords
Procarbazine (P) is an effective chemotherapeutic drug especially used in lymphoma treatment; however testicular toxicity is a limiting factor. Various ways of treatment were tried to preserve testicular function including hormonal treatment, antioxidant treatment, and sperm cryopreservation but resulted with low rates of satisfaction. Procarbazine is a well known agent causing sterility even in the first doses of chemotherapy. Antioxidants such as N acetylcysteine and ascorbate have been used for protective purposes and were very successful. Melatonin (M) is another powerful antioxidant and we aimed to use M for the protection of P induced testicular toxicity in this study. Procarbazine was given peroral by gavage once a week at a dose of 62.5 mg/kg/week for 4 weeks (total dose: 250 mg/kg) (P group) and in procarbazine + melatonin (PM) group, 10 mg/kg melatonin was intraperitoneally administered daily for five days a week for 4 weeks (total 20 days). The experiment ended at day 90. In the P and PM groups the testicle width, length, and weight, sperm A and sperm AB properties (Sperm A: sperms straight line progressive, Sperm B: sperms straight slow progressive, Sperm AB: Sperm A + Sperm B), spermatogonia, Sertoli cells, seminiferous tubule, and germinative layer thickness were lowered as compared with the control group. However, there were no significant differences between the P and PM groups in regard to these parameters. Melatonin preserved Sertoli cell and spermatogonia function. The testosterone and folliclestimulating hormone (FSH) levels were also preserved. Melatonin significantly decreased malondialdehyde (MDA) levels and preserved the antioxidant enzyme levels such as glutathione peroxidase (GPx) and nitrite nitrate (NO 2 =NO3 ). Melatonin may protect testicular functions in P treated patients and is open to consideration during chemotherapy since it appears to be without any side effects.
Antioxidant, melatonin, procarbazine, sterility, testicular toxicity History Received 16 December 2013 Revised 27 April 2014 Accepted 30 April 2014 Published online 20 August 2014
Abbreviations: P: procarbazine; GnRH: gonadotropin–releasing hormone; M: melatonin; PM: procarbazine + melatonin; FSH: follicle-stimulating hormone; LH: luteinizing hormone; MDA: malondialdehyde; CuZn-SOD: Cu Zn superoxide dismutase; GPx: glutathione peroxidase NO 2 =NO3 nitrite nitrate; SF: serum physiologic
Introduction Procarbazine (P) is an effective drug used for the treatment of many cancers especially for Hodgkin’s lymphoma. However, P induces a high rate of testicular damage even after a single dose that is manifested by the failure of spermatogenesis which is a limiting factor for the use of the drug [Spivack 1974]. Although the survival rates of cancer patients increase, the patients face high rates of sterility in their later life. Thus, protecting the patient from the side effects of P has much significance.
*Address correspondence to Bilal Fırat Alp, Gulhane Military Medical Academy, Department of Urology, Etlik 06018, Ankara, Turkey. Tel: +90 312 3045605. E-mail: [email protected]
Suppressing the hormonal axis and keeping the testes quiescence during chemotherapy or radiotherapy has been used as a protective approach. Gonadotropin–releasing hormone (GnRH)-agonists, GnRH antagonists, and antiandrogen drugs have been used to decrease the side effects and stimulate the recovery of spermatogenesis but they have had limited success [Kangasniemi et al. 1995; Meistrich et al. 1999]. While semen cryopreservation is a method it cannot be used for children. Another mechanism of spermiotoxicity caused by P was reported to be via bioactivation of P to an alkylating intermediate along with oxidation [Yost et al. 1985] and the formation of methyl radical [Moloney and Prough 1983] or other radicals [Sinha 1984] during the metabolism of P. Thus, in an injury mostly induced by oxidative damage, protection with antioxidant agents can be considered. From that point
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of view, P co-administered with powerful antioxidants such as N-acetylcysteine and sodium ascorbate decreased spermiotoxicity [Horstman et al. 1987]. Melatonin (M), produced by the pineal gland, is a well-known antioxidant and free radical scavenger [Reiter et al. 2000; Tan et al. 2000]. Melatonin is a very effective defender molecule against many carcinogens causing oxidative damage [Karbownik et al. 2000; Korkmaz et al. 2009; Qi et al. 2000]. Therefore, we investigated the protective effects of melatonin against the spermiotoxicity caused by P.
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Results Of the 21 animals used in the study 19 survived all treatments. The two animals that expired during the experimental period most likely succumbed to chemotherapy toxicity. Procarbazine caused severe damage to the testes. In the P and procarbazine + melatonin (PM) groups, the testicle width, length, and weight, sperm AB, sperm A, spermatogonia, Sertoli cells, seminiferous tubule, and germinative layer thickness were lowered as compared with the control group. However, there were no significant differences between the P and PM groups with regard to these parameters. Spermatogonia levels in the PM group were higher than the P group but it was not statistically significant (Table 1).
The serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone levels are presented in Table 2. The FSH levels increased and testosterone levels decreased in the P group as compared with the control group but it was not significant. Additionally, serum testosterone levels increased in the PM group as compared with the control group (Table 2). The products of lipid peroxidation and antioxidant enzyme levels are presented in Table 2. This included malondialdehyde (MDA), Cu Zn superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx), and nitrite nitrate (NO 2 =NO3 ). The level of MDA in the P group was significantly higher than the PM and C groups demonstrating tissue damage (p50.05). In comparison the level of MDA was significantly less in the PM group as compared with the P group showing the protective effects of melatonin (p50.05). The GPx levels were significantly decreased in the P group when compared with the control group (p ¼ 0.037). However, the levels did not significantly change in the PM group demonstrating the protective effect of melatonin (p50.05). The CuZnSOD and NO 2 =NO3 levels were increased in the P group when compared with the control group but it was only significant for NO 2 =NO3 , p ¼ 0.016 as illustrated in Table 2. As shown in Figure 1, when compared to the P group, thicknesses of layers were increased in the PM group. The architecture of the seminiferous tubules was observed
Table 1. The comparison of the groups in regard to spermiotoxicity parameters.
Groups Testicle width (cm) Testicle length (cm) Testicle weight (g) Rat weight (g) Sperm AB (106) Sperm A (106) Spermatogonia Sertoli cell Seminiferous tubule thickness (mm) Germinative layer thickness (mm)
Procarbazine (P) (n:8)
Procarbazine + Melatonin (PM) (n:6)
Control (C) (n:7)
1 ± 0.18 (0.5–1.1) 1.7 ± 0.09 (1.6–1.9) 0.65 ± 0.08 (0.5–0.8) 366 ± 35.2 (300–403) 2 ± 1.24 (0–4) 0 ± 1.4 (0–4) 9.65 ± 2.07 (6.67–14) 5.6 ± 1.4 (3.67–6.6) 148.95 ± 18.74 (119.70–178.90)
1 ± 0.04 (0.9–1) 1.65 ± 0.1 (1.5–1.7) 0.63 ± 0.17 (0.45–0.98) 334.5 ± 36.17 (314–403) 1 ± 1.87 (0–5) 0 ± 0.4 (0–1) 13.61 ± 10.03 (7.02–35.3) 5 ± 4.4 (1.6–14.5) 143.20 ± 40.02 (137.70–240.70)
1.3 ± 0.1 (1.1–1.4) 2.1 ± 0.07 (2–2.2) 1.9 ± 0.08 (1.7–1.9) 384 ± 41.1 (313–426) 11 ± 7 (5–26) 3 ± 1.5 (2–6) 57.6 ± 4.2 (52.1–65.6) 20 ± 2.9 (18.9–27.6) 252.30 ± 32.20 (219.20–295.20)
45.7 ± 8.4 (31.6–57.8)
43.5 ± 17.9 (36.5–85.5)
76.2 ± 7.8 (70–90.4)
p *0.001 *0.001 *0.001
y0.002 y0.002 y0.003
*0.015 *0.012 *0.001 *0.001 *0.01
y0.003 y0.002 y0.003 y0.003 y0.01
*0.001
y0.022
Data are presented as median ± standard deviation. p values: *: P vs. C; y: PM vs. C; #: P vs. PM. Sperm A: sperms straight line progressive; Sperm B: sperms straight slow progressive; Sperm AB: Sperm A + Sperm B.
Table 2. The hormonal, lipid peroxidation product, and antioxidant enzyme status of the groups.
Groups FSH (mIU/ml) LH (mIU/ml) Testosterone (ng/dl) MDA (nmol/ml) CuZnSOD (U/ml) GPx (U/ml) NO2/NO3 (mmol/ml)
Procarbazine (P) (n:8)
Procarbazine + Melatonin (PM) (n:6)
Control (C) (n:7)
0.76 ± 0.24 (0.43–1.1) 0.001 ± 0.007 (0.001–0.02) 79.58 ± 27.5 (59.3–140.5) 4.2 ± 0.4 (3.6–4.7) 1.8 ± 0.3 (1.5–2.2) 6.9 ± 0.5 (6.5–8.1) 0.17 ± 0.02 (0.12–0.19)
0.58 ± 0.13 (0.48–0.83) 0.001 ± 0 (0.001–0.001) 131.84 ± 65.78 (62.68–233.4) 3.5 ± 0.5 (2.5–4.06) 1.7 ± 0.4 (0.9–2.0) 8.2 ± 0.7 (7.04–9.1) 0.13 ± 0.02 (0.09–0.17)
0.5 ± 0.18 (0.17–0.7) 0.001 ± 0.04 (0.001–0.11) 109.9 ± 57.9 (38.9–213.3) 3.6 ± 0.8 (2.2–4.2) 1.45 ± 0.4 (0.9–2) 8.4 ± 1.1 (6.1–9.5) 0.12 ± 0.03 (0.09–0.16)
p
*0.028 *0.037 #0.010 *0.016 #0.036
Data are presented as median ± standard deviation. p Values: #: P vs. PM; *: P vs. C; y: PM vs. C. FSH; follicle-stimulating hormone; LH: luteinizing hormone; MDA: malondialdehyde; CuZn-SOD: Cu Zn superoxide dismutase; GPx: glutathione peroxidase; NO 2 =NO3 : nitrite nitrate.
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DOI: 10.3109/19396368.2014.930212
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Figure 1. Representative pathologic figures of the study groups demonstrating the effects of procarbazine and melatonin with comparison of the normal testes. A1 and 2) Most of the seminiferous tubules show severe injury and lack sperm production in the procarbazine treated group (HEx200, HEx400). B1 and 2) Although the interstitium looks congested, seminiferous tubules are structurally intact in the melatonin treated group (HEx100, HEx400). C1 and 2) Normal testes parenchyma from the control group for comparison (HEx100, HEx400).
to be more protected in the PM group. Germinative cells were increased in the PM group compared to the P group. Morphology, localization, and maturation of germinative cells were closer to normal in the PM group then the P group. Similarly, morphology and localization of Sertoli cells were better in the PM group then the P group. Although in the histopathological evaluation of the PM group suggested protection compared to the P group, there was no statistically significant difference between the P and PM groups.
Discussion Although cancer therapy survival rates are increasing, a late effect of chemotherapy such as sterility is still a big problem among survivors. Procarbazine has a high risk of gonadal dysfunction. In our study, M preserved spermatogonia. Histopathologically, the morphology of Sertoli cells and seminiferous and germinative layers were much healthier in the M supplemented group. The testosterone and FSH levels were also preserved. The antioxidant status was clearly preserved. Preserving gonadal function is a significant issue among cancer survivors. Great effort was spent to protect the patients from the harmful effects of P. In recent years, hormonal therapy, semen cryopreservation, and antioxidant treatment were all tried and experienced limitations. Semen cryopreservation is a well known treatment among older patients. However, conveying the understanding about disease and fertility preservation, obtaining a semen sample of sufficient volume, and sperm content are limitations among adolescent patients. Since chemotherapy affects mainly rapidly dividing cells, the basis of hormonal treatment depends on suppressing and manipulating germ cells to rest for minimizing the toxic effects of chemotherapy. This technique has been
successful in animals [Kurdoglu et al. 1994]. However, there is no success in clinical trials [Thomson et al. 2002]. Moreover, the success of the hormonal treatment depends on the suppression of the gonadotrophin axis and in children is low, because the pioneer studies on prepubertal animals evaluating the proliferation of germ cells might be gonadotrophin independent [Kelnar et al. 2002]. Procarbazine related testicular damage affects Sertoli cell nurturing the developing germ cells and Leydig cells producing testosterone. Melatonin preserved both the testosterone levels and Sertoli cells in the PM group as compared with the P group but it was not statistically significant. The increase in testosterone levels in the M supplemented group is consistent with its protection of Leydig cells. This situation reflects that spermatogenesis may be stimulated by testosterone’s effect on the protected spermatogonia. This might be due to the low number of rats in the groups. The seminiferous epithelium of the testes is the most sensitive to chemotherapy resulting with oligospermia or azoospermia. Although the exact mechanism is unclear, the damage kills the proliferating germ cells at any stage and also the stem cells. In our study, the P treated groups showed devastating damage in the number and velocity of sperm AB and A, Sertoli cells, spermatogonias, and the thickness of seminiferous layer and germinative layers. Melatonin supplementation protected the levels of spermatogonia and also seminiferous and germinative layers morphologically but there was no significant difference due to the low number of animals in the study groups. Chemotherapeutic agents are known to lower testis dimensions [Atessahin et al. 2006]. Atessahin et al. [2006] reported that the protective effect of melatonin on testis dimensions and weight induced by cisplatin induced testicular damage. Procarbazine lowered the testicle dimensions, weight, and also the weight of the rat as compared with the control group. However, there was no significant difference.
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There was also no difference between groups regarding these parameters reflecting that melatonin has no protective effect. Horstman et al. [1987] clearly demonstrated that antioxidant co-administration such as acetylcysteine and ascorbate decreased the spermiotoxicity of P without changing its anticancer efficacy. The mechanism of gonadal protection was indicated as either a reduction of oxidized intermediates or an interception of electrophilic alkylating intermediates by protective nucleophilic biomolecules such as glutathione. In conclusion, the authors suggested that the oxidation of the methylazoxy P isomer at the benzylic position to produce an electrophilic intermediate is the most likely mechanism of P spermiotoxicity. Also, chemotherapy agents cause tissue damage resulting with oxidant damage [Horstman et al. 1987]. In our study, M administration decreased MDA levels and preserved GPx and NO 2 =NO3 levels. Horstman et al. [1987] demonstrated that acetylcysteine and ascorbate protect spermatids against P toxicity. However, we found that M protects mostly spermatogonia function rather than other types of spermatogenic cells. Although the spermatogonia, stem cell of spermatogenesis, is the cell in the first side of the blood-testis barrier that may face oxidants in the first line, it was found more stable and more resistant to the oxidant damage than other cells in the spermatogenesis. Testicular toxicity of P is believed to reflect the free radicals generated in testicular tissue. This situation is clearly demonstrated in our study with increased levels of MDA in the P treated group presenting increased levels of oxidant status. Spermatogenic cells at all stages are rich in polyunsaturated fatty acids that make them susceptible to free oxygen radical attack. This situation results with a decreased sperm count, motility, sperm viability, and severe morphological changes. The underlying mechanism might be via a rapid loss of intracellular ATP. Melatonin is known to lower mitochondrial protein damage, improve electron transport chain activity, and reduce mtDNA damage. Melatonin also improves mitochondrial respiration and increases ATP synthesis under normal and stressful conditions. Thus, M preserves sperm function by sparing mitochondrial integrity, preventing alterations in mitochondrial membrane potential which would have otherwise triggered an apoptotic cascade [Leon et al. 2005]. In our study, we observed an enormous antioxidant effect that was presented directly with the preserved levels of MDA, GPx, CuZnSOD, and NO 2 =NO3 . The possible mechanisms of which M modulates the activity of enzymatic and nonenzymatic antioxidants were suggested as regulation of antioxidant enzyme gene expression and inactivation of nuclear RORalpha M receptor via interaction with calmodulin [Mayo 2002; Toma´s-Zapico and Coto-Montes 2005]. In conclusion, oxidant damage is not the only mechanism in P related gonadal toxicity. However, M preserved Sertoli cell and spermatogonia function, the testosterone and FSH levels, and antioxidant enzyme levels. All these results demonstrate a limited preserving effect of M. We think that the limitation of this study is the number of subjects in the study groups. Thus, increasing the number of subjects and
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administering higher doses of M in a further study may present clearer and statistically significant results.
Materials and Methods Animals and diets A total of 21 adult, male Wistar–Albino rats weighing approximately 250–300 g were used for the experiment. All rats were maintained in a controlled temperature at 21–22 C, 45–55% humidity environment with a regular light and dark period (12 h light, 12 h dark), and fed with 24% protein added commercial rat diet ad libitum. The rats were randomly distributed into three groups: control (C) group (n: 7), procarbazine (P) treated group (n: 8), and procarbazine + melatonin (PM) treated group (n: 6, 2 rats from this group died during the experimental period). In the C group, the rats were fed with 0.5 mL serum physiologic (SF) peroral once a w similar with the other groups. In the P and PM treated groups, P was applied peroral by gavage once a w at a dose of 62.5 mg/kg/w for 4 w (total dose: 250 mg/kg). In the PM group, 10 mg/kg M was administered intraperitoneally daily at the same time (8 in the evening) in order to avoid circadian rhyme for 5 days per w for 4 w (total 20 d). The Experimentation Ethics Committee of our institution approved the experimental procedures of the study. Preparation of drugs Melatonin (Sigma Chemical, St Louis, MO) was administered intraperitoneally at a dose of 10 mg/kg dissolved in 0.5 ml saline under sterile conditions for 5 days per w for 4 weeks [Guven et al. 2011]. Procarbazine (NatulanÕ Sigma Tau, France) was administered peroral by gavage at a dose of 62.5 mg/kg/w for 4 w dissolved in 1 ml saline under sterile conditions once a w for 4 w [Parchuri et al. 1993]. Study design The rats were maintained for 90 d after the first dose of P as mentioned in the literature [Gocmen et al. 2001]. After the end of the study process, the rats were anesthetized with isofluorane gas anesthesia and blood removed by cardiac puncture into heparinized tubes for hormonal and antioxidant enzyme assays. The serum for determining antioxidant enzyme levels were placed into separate tubes and frozen in liquid nitrogen. The tissues were kept frozen at 80 C until assayed. The testis and epididymis were carefully dissected out, all fat and blood was trimmed. The right epididymis was removed and rinsed immediately in a plate that was filled with 2 ml of SF (for standardization of the ejaculate volume). After a 5 min incubation of the semen in SF, 50 microliters of this solution was added to a 450 microliter saline for a final concentration of 500 microliters for analyses. Fifty microliters from the final specimen was then placed into the sperm analyzer for analyzing. The left testis, epididymis, and seminal vesicle were removed and after washing in normal saline, their weight and volume were measured. After these measurements, the testis was placed into formaldehyde for histopathologic analyses.
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Sperm motility and morphology The motility and morphology was analyzed with the BiolaÕ automatic sperm analyzer model SFA-500 (Biola SFA-500, Russia) and the data was calculated with its original software Sa8 version 8.1194. The device is produced to analyze sperm of both humans and animals. The software was calibrated for use with rat sperm to analyze rats. For motility analyses, 50 microliters of the specimen was loaded into the analyzers special cuvette. Then the cuvette was placed into the analyzer for sperm counting. The analyzer automatically measured the sperm parameters including sperm concentration per ml and sperm motility (fast linear motility, slow linear motility).
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Biochemical analysis GPx activity measurement GPx activities in serum were measured by the method described previously [Sun et al. 1988]. The reaction mixture was 50 mmol/L tris buffer, pH 7.6 containing 1 mmol/L of Na2EDTA, 2 mmol/L of reduced glutathione (GSH), 0.2 mmol/L of NADPH, 4 mmol/L of sodium azide, and 1000 U of glutathione reductase (GR). Fifty ml of plasma and 950 ml of reaction mixture were mixed and incubated for 5 min at 37 C. Then the reaction was initiated with 8.8 mmol/L H2O2 and the decrease in NADPH absorbance was followed at 340 nm for 3 min. GPx activity was expressed in U/ml [Lawrence and Burk 2012]. CuZn-SOD activity measurement CuZn-SOD activity in serum was determined according to the method of Sun et al. [1988]. It is based on the inhibition of nitroblue tetrazolium (NBT) reduction by the xanthine/ xanthine oxidase system as a superoxide generator. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. CuZn-SOD activity was expressed in U/ml [Sun et al. 1988].
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After incubating for 10 min, the absorbance was measured spectrometrically at 540 nm. The nitrite/nitrate concentrations in the samples were calculated from a standard curve [Tracey et al. 1995]. Serum FSH, LH, and testosterone analyses used standard immunoassay methods. Serum follicle-stimulating hormone, LH, and testosterone levels were measured by ADVIA Centaur XP Immunoassay System using its kits (Siemens Healthcare Diagnostics Ltd., Erlangen, Germany). Histopathological analyses The testis specimens were fixed in 10% buffered formaldehyde. After routine pathological procedure, 5 micron section slides were prepared. Slides were stained with hematoxylen-eosine (HE). The slides were examined by a specialized pathologist blinded to all groups. Seminiferous tubules were counted and examined on a light microscope (40X, HPF). Forty-five seminiferous tubules were examined for each rat. Sertoli cells and spermatogonias were counted in each seminiferous tubule. Germinative layer and seminiferous tubule width and length were calculated. And then median levels of all 45 counts were calculated. Statistical analysis Results were expressed as the mean plus/minus standard error of the mean (mean ± S.E.M.). Numeric data were analyzed first using the Kruskal–Wallis test to determine differences between the groups, then the Mann–Whitney U-test was employed to analyze two groups consecutively. p values of 50.05 were regarded as significant. SPSS for windows program version 10.0 was used for the statistical analyses.
Declaration of interest The authors report no declarations of interest.
Author contributions MDA level measurement Serum MDA levels were determined by the method described previously. After the reaction of thiobarbituric acid with MDA, the reaction product was extracted in butanol and was measured spectrofluorometrically (excitation:532 nm, emission: 553 nm, slit 10 nm). After the reaction of thiobarbituric acid with MDA, the reaction product was measured spectrometrically. Tetramethoxy propane solution was used as standard. The MDA levels of serum were expressed as nmol/ml [Ohkawa et al. 1979]. NO 2 =NO3 level measurement Serum NO concentrations were determined by 2 =NO3 using the Griess reaction according to Tracey et al. [1995]. The reaction mixture consisted of reduced nicotineamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and nitrate reductase. After incubation of serum samples with reaction mixture, Griess reagent (a 1:1 mixture of 1% sulfanilamide in 5% H3PO4 and 0.1% N-[1-naphtyl]-ethylenediamine) was added to the samples.
All authors contributed extensively to the work presented in this paper. Conceived and designed the experiments: VK; Performed the experiments: VK, BFA, YP; Analyzed the data: VK, MG; Contributed reagents/materials/analysis tools: EOA, OE, NY; Pathologic examinations: NY; Wrote the manuscript: VK, BFA, MG, OB, EOA, NK, MS, NY; Revised the manuscript: VK, EM, BFA, MS. All authors approved the revisions to the manuscript.
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Kangasniemi, M., Wilson, G., Parchuri, N., Huhtaniemi, I. and Meistrich, M.L. (1995) Rapid protection of rat spermatogenic stem cells against procarbazine by treatment with a gonadotropin-releasing hormone antagonist (Nal-Glu) and an antiandrogen (flutamide). Endocrinology 136:2881–2888. Karbownik, M., Tan, D.X. and Reiter, R.J. (2000) Melatonin reduces the oxidation of nuclear DNA and membrane lipids induced by the carcinogen delta-aminolevulinic acid. Int J Cancer 88:7–11. Kelnar, C.J., McKinnell, C., Walker, M., Morris, K.D., Wallace, W.H., Saunders, P.T., et al. (2002) Testicular changes during infantile ‘quiescence’ in the marmoset and their gonadotrophin dependence: a model for investigating susceptibility of the prepubertal human testis to cancer therapy? Hum Reprod 17:1367–1378. Korkmaz, A., Reiter, R.J., Topal, T., Manchester, L.C., Oter, S. and Tan, D.X. (2009) Melatonin: an established antioxidant worthy of use in clinical trials. Mol Med 15:43–50. Kurdoglu, B., Wilson, G., Parchuri, N., Ye, W.S. and Meistrich, M.L. (1994) Protection from radiation-induced damage to spermatogenesis by hormone treatment. Radiat Res 139:97–102. Lawrence, R.A. and Burk, R.F. (2012) Glutathione peroxidase activity in selenium-deficient rat liver. 1976. Biochem Biophys Res Commun 425:503–509. Leon, J., Acuna-Castroviejo, D., Escames, G., Tan, D.X. and Reiter, R.J. (2005) Melatonin mitigates mitochondrial malfunction. J Pineal Res 38:1–9. Mayo, J.C., Sainz, R.M., Antoli, I., Herrera, F., Martin, V. and Rodriguez, C. (2002) Melatonin regulation of antioxidant enzyme gene expression. Cell Mol Life Sci 59:1706–1713. Meistrich, M.L., Wilson, G. and Huhtaniemi, I. (1999) Hormonal treatment after cytotoxic therapy stimulates recovery of spermatogenesis. Cancer Res 59:3557–3560. Moloney, S.J. and Prough, R.A. (1983) Studies on the pathway of methane formation from procarbazine, a 2-methylbenzylhydrazine derivative, by rat liver microsomes. Arch Biochem Biophys 221:577–584. Ohkawa, H., Ohishi, N. and Yagi, K. (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95: 351–358.
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Parchuri, N., Wilson, G. and Meistrich, M.L. (1993) Protection by gonadal steroid hormones against procarbazine-induced damage to spermatogenic function in LBNF1 hybrid rats. J Androl 14:257–266. Qi, W., Reiter, R.J., Tan, D.X., Manchester, L.C., Siu, A.W. and Garcia, J.J. (2000) Increased levels of oxidatively damaged DNA induced by chromium(III) and H2O2: protection by melatonin and related molecules. J Pineal Res 29:54–61. Reiter, R.J., Tan, D.X., Qi, W., Manchester, L.C., Karbownik, M. and Calvo, J.R. (2000) Pharmacology and physiology of melatonin in the reduction of oxidative stress in vivo. Biol Signals Recept 9: 160–171. Sinha, B.K. (1984) Metabolic activation of procarbazine. Evidence for carbon-centered free-radical intermediates. Biochem Pharmacol 33: 2777–2781. Spivack, S.D. (1974) Drugs 5 years later: procarbazine. Ann Intern Med 81:795–800. Sun, Y., Oberley, L.W. and Li, Y. (1988) A simple method for clinical assay of superoxide dismutase. Clin Chem 34:497–500. Tan, D.X., Manchester, L.C., Reiter, R.J., Qi, W.B., Karbownik, M. and Calvo, J.R. (2000) Significance of melatonin in antioxidative defense system: reactions and products. Biol Signals Recept 9: 137–159. Toma´s-Zapico, C. and Coto-Montes, A. (2005) A proposed mechanism to explain the stimulatory effect of melatonin on antioxidative enzymes. J Pineal Res 39:99–104. Thomson, A.B., Anderson, R.A., Irvine, D.S., Kelnar, C.J., Sharpe, R.M. and Wallace, W.H. (2002) Investigation of suppression of the hypothalamic-pituitary-gonadal axis to restore spermatogenesis in azoospermic men treated for childhood cancer. Hum Reprod 17: 1715–1723. Tracey, W.R., Tse, J. and Carter, G. (1995) Lipopolysaccharide-induced changes in plasma nitrite and nitrate concentrations in rats and mice: pharmacological evaluation of nitric oxide synthase inhibitors. J Pharmacol Exp Ther 272:1011–1015. Yost, G.S., Horstman, M.G., el Walily, A.F., Gordon, W.P. and Nelson, S.D. (1985) Procarbazine spermatogenesis toxicity: deuterium isotope effects point to regioselective metabolism in mice. Toxicol Appl Pharmacol 80:316–322.
Notice of correction: The surname of the sixth author has been corrected since the original online publication date of August 20, 2014.
RESEARCH COMMUNICATION
The effect of melatonin on procarbazine induced testicular toxicity on rats Bilal Fırat Alp1*, Vural Kesik2, Ercan Malkoc¸1, Nuri Yig˘it3, Mehmet Saldır4, Og˘uzhan Babacan2, Emin O¨zgu¨r Akgu¨l5, Yavuz Poyrazoglu6, Nadir Korkmazer4, Mustafa Gulgun4, and Onur Erdem7
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1
Department of Urology, 2Pediatric Hematology and Oncology, 3Pathology, 4Pediatrics, 5Biochemistry, 6Surgery, and 7Toxicology, Gulhane Military Medical Faculty, Ankara, Turkey Abstract
Keywords
Procarbazine (P) is an effective chemotherapeutic drug especially used in lymphoma treatment; however testicular toxicity is a limiting factor. Various ways of treatment were tried to preserve testicular function including hormonal treatment, antioxidant treatment, and sperm cryopreservation but resulted with low rates of satisfaction. Procarbazine is a well known agent causing sterility even in the first doses of chemotherapy. Antioxidants such as N acetylcysteine and ascorbate have been used for protective purposes and were very successful. Melatonin (M) is another powerful antioxidant and we aimed to use M for the protection of P induced testicular toxicity in this study. Procarbazine was given peroral by gavage once a week at a dose of 62.5 mg/kg/week for 4 weeks (total dose: 250 mg/kg) (P group) and in procarbazine + melatonin (PM) group, 10 mg/kg melatonin was intraperitoneally administered daily for five days a week for 4 weeks (total 20 days). The experiment ended at day 90. In the P and PM groups the testicle width, length, and weight, sperm A and sperm AB properties (Sperm A: sperms straight line progressive, Sperm B: sperms straight slow progressive, Sperm AB: Sperm A + Sperm B), spermatogonia, Sertoli cells, seminiferous tubule, and germinative layer thickness were lowered as compared with the control group. However, there were no significant differences between the P and PM groups in regard to these parameters. Melatonin preserved Sertoli cell and spermatogonia function. The testosterone and folliclestimulating hormone (FSH) levels were also preserved. Melatonin significantly decreased malondialdehyde (MDA) levels and preserved the antioxidant enzyme levels such as glutathione peroxidase (GPx) and nitrite nitrate (NO 2 =NO3 ). Melatonin may protect testicular functions in P treated patients and is open to consideration during chemotherapy since it appears to be without any side effects.
Antioxidant, melatonin, procarbazine, sterility, testicular toxicity History Received 16 December 2013 Revised 27 April 2014 Accepted 30 April 2014 Published online 20 August 2014
Abbreviations: P: procarbazine; GnRH: gonadotropin–releasing hormone; M: melatonin; PM: procarbazine + melatonin; FSH: follicle-stimulating hormone; LH: luteinizing hormone; MDA: malondialdehyde; CuZn-SOD: Cu Zn superoxide dismutase; GPx: glutathione peroxidase NO 2 =NO3 nitrite nitrate; SF: serum physiologic
Introduction Procarbazine (P) is an effective drug used for the treatment of many cancers especially for Hodgkin’s lymphoma. However, P induces a high rate of testicular damage even after a single dose that is manifested by the failure of spermatogenesis which is a limiting factor for the use of the drug [Spivack 1974]. Although the survival rates of cancer patients increase, the patients face high rates of sterility in their later life. Thus, protecting the patient from the side effects of P has much significance.
*Address correspondence to Bilal Fırat Alp, Gulhane Military Medical Academy, Department of Urology, Etlik 06018, Ankara, Turkey. Tel: +90 312 3045605. E-mail: [email protected]
Suppressing the hormonal axis and keeping the testes quiescence during chemotherapy or radiotherapy has been used as a protective approach. Gonadotropin–releasing hormone (GnRH)-agonists, GnRH antagonists, and antiandrogen drugs have been used to decrease the side effects and stimulate the recovery of spermatogenesis but they have had limited success [Kangasniemi et al. 1995; Meistrich et al. 1999]. While semen cryopreservation is a method it cannot be used for children. Another mechanism of spermiotoxicity caused by P was reported to be via bioactivation of P to an alkylating intermediate along with oxidation [Yost et al. 1985] and the formation of methyl radical [Moloney and Prough 1983] or other radicals [Sinha 1984] during the metabolism of P. Thus, in an injury mostly induced by oxidative damage, protection with antioxidant agents can be considered. From that point
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of view, P co-administered with powerful antioxidants such as N-acetylcysteine and sodium ascorbate decreased spermiotoxicity [Horstman et al. 1987]. Melatonin (M), produced by the pineal gland, is a well-known antioxidant and free radical scavenger [Reiter et al. 2000; Tan et al. 2000]. Melatonin is a very effective defender molecule against many carcinogens causing oxidative damage [Karbownik et al. 2000; Korkmaz et al. 2009; Qi et al. 2000]. Therefore, we investigated the protective effects of melatonin against the spermiotoxicity caused by P.
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Results Of the 21 animals used in the study 19 survived all treatments. The two animals that expired during the experimental period most likely succumbed to chemotherapy toxicity. Procarbazine caused severe damage to the testes. In the P and procarbazine + melatonin (PM) groups, the testicle width, length, and weight, sperm AB, sperm A, spermatogonia, Sertoli cells, seminiferous tubule, and germinative layer thickness were lowered as compared with the control group. However, there were no significant differences between the P and PM groups with regard to these parameters. Spermatogonia levels in the PM group were higher than the P group but it was not statistically significant (Table 1).
The serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone levels are presented in Table 2. The FSH levels increased and testosterone levels decreased in the P group as compared with the control group but it was not significant. Additionally, serum testosterone levels increased in the PM group as compared with the control group (Table 2). The products of lipid peroxidation and antioxidant enzyme levels are presented in Table 2. This included malondialdehyde (MDA), Cu Zn superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx), and nitrite nitrate (NO 2 =NO3 ). The level of MDA in the P group was significantly higher than the PM and C groups demonstrating tissue damage (p50.05). In comparison the level of MDA was significantly less in the PM group as compared with the P group showing the protective effects of melatonin (p50.05). The GPx levels were significantly decreased in the P group when compared with the control group (p ¼ 0.037). However, the levels did not significantly change in the PM group demonstrating the protective effect of melatonin (p50.05). The CuZnSOD and NO 2 =NO3 levels were increased in the P group when compared with the control group but it was only significant for NO 2 =NO3 , p ¼ 0.016 as illustrated in Table 2. As shown in Figure 1, when compared to the P group, thicknesses of layers were increased in the PM group. The architecture of the seminiferous tubules was observed
Table 1. The comparison of the groups in regard to spermiotoxicity parameters.
Groups Testicle width (cm) Testicle length (cm) Testicle weight (g) Rat weight (g) Sperm AB (106) Sperm A (106) Spermatogonia Sertoli cell Seminiferous tubule thickness (mm) Germinative layer thickness (mm)
Procarbazine (P) (n:8)
Procarbazine + Melatonin (PM) (n:6)
Control (C) (n:7)
1 ± 0.18 (0.5–1.1) 1.7 ± 0.09 (1.6–1.9) 0.65 ± 0.08 (0.5–0.8) 366 ± 35.2 (300–403) 2 ± 1.24 (0–4) 0 ± 1.4 (0–4) 9.65 ± 2.07 (6.67–14) 5.6 ± 1.4 (3.67–6.6) 148.95 ± 18.74 (119.70–178.90)
1 ± 0.04 (0.9–1) 1.65 ± 0.1 (1.5–1.7) 0.63 ± 0.17 (0.45–0.98) 334.5 ± 36.17 (314–403) 1 ± 1.87 (0–5) 0 ± 0.4 (0–1) 13.61 ± 10.03 (7.02–35.3) 5 ± 4.4 (1.6–14.5) 143.20 ± 40.02 (137.70–240.70)
1.3 ± 0.1 (1.1–1.4) 2.1 ± 0.07 (2–2.2) 1.9 ± 0.08 (1.7–1.9) 384 ± 41.1 (313–426) 11 ± 7 (5–26) 3 ± 1.5 (2–6) 57.6 ± 4.2 (52.1–65.6) 20 ± 2.9 (18.9–27.6) 252.30 ± 32.20 (219.20–295.20)
45.7 ± 8.4 (31.6–57.8)
43.5 ± 17.9 (36.5–85.5)
76.2 ± 7.8 (70–90.4)
p *0.001 *0.001 *0.001
y0.002 y0.002 y0.003
*0.015 *0.012 *0.001 *0.001 *0.01
y0.003 y0.002 y0.003 y0.003 y0.01
*0.001
y0.022
Data are presented as median ± standard deviation. p values: *: P vs. C; y: PM vs. C; #: P vs. PM. Sperm A: sperms straight line progressive; Sperm B: sperms straight slow progressive; Sperm AB: Sperm A + Sperm B.
Table 2. The hormonal, lipid peroxidation product, and antioxidant enzyme status of the groups.
Groups FSH (mIU/ml) LH (mIU/ml) Testosterone (ng/dl) MDA (nmol/ml) CuZnSOD (U/ml) GPx (U/ml) NO2/NO3 (mmol/ml)
Procarbazine (P) (n:8)
Procarbazine + Melatonin (PM) (n:6)
Control (C) (n:7)
0.76 ± 0.24 (0.43–1.1) 0.001 ± 0.007 (0.001–0.02) 79.58 ± 27.5 (59.3–140.5) 4.2 ± 0.4 (3.6–4.7) 1.8 ± 0.3 (1.5–2.2) 6.9 ± 0.5 (6.5–8.1) 0.17 ± 0.02 (0.12–0.19)
0.58 ± 0.13 (0.48–0.83) 0.001 ± 0 (0.001–0.001) 131.84 ± 65.78 (62.68–233.4) 3.5 ± 0.5 (2.5–4.06) 1.7 ± 0.4 (0.9–2.0) 8.2 ± 0.7 (7.04–9.1) 0.13 ± 0.02 (0.09–0.17)
0.5 ± 0.18 (0.17–0.7) 0.001 ± 0.04 (0.001–0.11) 109.9 ± 57.9 (38.9–213.3) 3.6 ± 0.8 (2.2–4.2) 1.45 ± 0.4 (0.9–2) 8.4 ± 1.1 (6.1–9.5) 0.12 ± 0.03 (0.09–0.16)
p
*0.028 *0.037 #0.010 *0.016 #0.036
Data are presented as median ± standard deviation. p Values: #: P vs. PM; *: P vs. C; y: PM vs. C. FSH; follicle-stimulating hormone; LH: luteinizing hormone; MDA: malondialdehyde; CuZn-SOD: Cu Zn superoxide dismutase; GPx: glutathione peroxidase; NO 2 =NO3 : nitrite nitrate.
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Figure 1. Representative pathologic figures of the study groups demonstrating the effects of procarbazine and melatonin with comparison of the normal testes. A1 and 2) Most of the seminiferous tubules show severe injury and lack sperm production in the procarbazine treated group (HEx200, HEx400). B1 and 2) Although the interstitium looks congested, seminiferous tubules are structurally intact in the melatonin treated group (HEx100, HEx400). C1 and 2) Normal testes parenchyma from the control group for comparison (HEx100, HEx400).
to be more protected in the PM group. Germinative cells were increased in the PM group compared to the P group. Morphology, localization, and maturation of germinative cells were closer to normal in the PM group then the P group. Similarly, morphology and localization of Sertoli cells were better in the PM group then the P group. Although in the histopathological evaluation of the PM group suggested protection compared to the P group, there was no statistically significant difference between the P and PM groups.
Discussion Although cancer therapy survival rates are increasing, a late effect of chemotherapy such as sterility is still a big problem among survivors. Procarbazine has a high risk of gonadal dysfunction. In our study, M preserved spermatogonia. Histopathologically, the morphology of Sertoli cells and seminiferous and germinative layers were much healthier in the M supplemented group. The testosterone and FSH levels were also preserved. The antioxidant status was clearly preserved. Preserving gonadal function is a significant issue among cancer survivors. Great effort was spent to protect the patients from the harmful effects of P. In recent years, hormonal therapy, semen cryopreservation, and antioxidant treatment were all tried and experienced limitations. Semen cryopreservation is a well known treatment among older patients. However, conveying the understanding about disease and fertility preservation, obtaining a semen sample of sufficient volume, and sperm content are limitations among adolescent patients. Since chemotherapy affects mainly rapidly dividing cells, the basis of hormonal treatment depends on suppressing and manipulating germ cells to rest for minimizing the toxic effects of chemotherapy. This technique has been
successful in animals [Kurdoglu et al. 1994]. However, there is no success in clinical trials [Thomson et al. 2002]. Moreover, the success of the hormonal treatment depends on the suppression of the gonadotrophin axis and in children is low, because the pioneer studies on prepubertal animals evaluating the proliferation of germ cells might be gonadotrophin independent [Kelnar et al. 2002]. Procarbazine related testicular damage affects Sertoli cell nurturing the developing germ cells and Leydig cells producing testosterone. Melatonin preserved both the testosterone levels and Sertoli cells in the PM group as compared with the P group but it was not statistically significant. The increase in testosterone levels in the M supplemented group is consistent with its protection of Leydig cells. This situation reflects that spermatogenesis may be stimulated by testosterone’s effect on the protected spermatogonia. This might be due to the low number of rats in the groups. The seminiferous epithelium of the testes is the most sensitive to chemotherapy resulting with oligospermia or azoospermia. Although the exact mechanism is unclear, the damage kills the proliferating germ cells at any stage and also the stem cells. In our study, the P treated groups showed devastating damage in the number and velocity of sperm AB and A, Sertoli cells, spermatogonias, and the thickness of seminiferous layer and germinative layers. Melatonin supplementation protected the levels of spermatogonia and also seminiferous and germinative layers morphologically but there was no significant difference due to the low number of animals in the study groups. Chemotherapeutic agents are known to lower testis dimensions [Atessahin et al. 2006]. Atessahin et al. [2006] reported that the protective effect of melatonin on testis dimensions and weight induced by cisplatin induced testicular damage. Procarbazine lowered the testicle dimensions, weight, and also the weight of the rat as compared with the control group. However, there was no significant difference.
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There was also no difference between groups regarding these parameters reflecting that melatonin has no protective effect. Horstman et al. [1987] clearly demonstrated that antioxidant co-administration such as acetylcysteine and ascorbate decreased the spermiotoxicity of P without changing its anticancer efficacy. The mechanism of gonadal protection was indicated as either a reduction of oxidized intermediates or an interception of electrophilic alkylating intermediates by protective nucleophilic biomolecules such as glutathione. In conclusion, the authors suggested that the oxidation of the methylazoxy P isomer at the benzylic position to produce an electrophilic intermediate is the most likely mechanism of P spermiotoxicity. Also, chemotherapy agents cause tissue damage resulting with oxidant damage [Horstman et al. 1987]. In our study, M administration decreased MDA levels and preserved GPx and NO 2 =NO3 levels. Horstman et al. [1987] demonstrated that acetylcysteine and ascorbate protect spermatids against P toxicity. However, we found that M protects mostly spermatogonia function rather than other types of spermatogenic cells. Although the spermatogonia, stem cell of spermatogenesis, is the cell in the first side of the blood-testis barrier that may face oxidants in the first line, it was found more stable and more resistant to the oxidant damage than other cells in the spermatogenesis. Testicular toxicity of P is believed to reflect the free radicals generated in testicular tissue. This situation is clearly demonstrated in our study with increased levels of MDA in the P treated group presenting increased levels of oxidant status. Spermatogenic cells at all stages are rich in polyunsaturated fatty acids that make them susceptible to free oxygen radical attack. This situation results with a decreased sperm count, motility, sperm viability, and severe morphological changes. The underlying mechanism might be via a rapid loss of intracellular ATP. Melatonin is known to lower mitochondrial protein damage, improve electron transport chain activity, and reduce mtDNA damage. Melatonin also improves mitochondrial respiration and increases ATP synthesis under normal and stressful conditions. Thus, M preserves sperm function by sparing mitochondrial integrity, preventing alterations in mitochondrial membrane potential which would have otherwise triggered an apoptotic cascade [Leon et al. 2005]. In our study, we observed an enormous antioxidant effect that was presented directly with the preserved levels of MDA, GPx, CuZnSOD, and NO 2 =NO3 . The possible mechanisms of which M modulates the activity of enzymatic and nonenzymatic antioxidants were suggested as regulation of antioxidant enzyme gene expression and inactivation of nuclear RORalpha M receptor via interaction with calmodulin [Mayo 2002; Toma´s-Zapico and Coto-Montes 2005]. In conclusion, oxidant damage is not the only mechanism in P related gonadal toxicity. However, M preserved Sertoli cell and spermatogonia function, the testosterone and FSH levels, and antioxidant enzyme levels. All these results demonstrate a limited preserving effect of M. We think that the limitation of this study is the number of subjects in the study groups. Thus, increasing the number of subjects and
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administering higher doses of M in a further study may present clearer and statistically significant results.
Materials and Methods Animals and diets A total of 21 adult, male Wistar–Albino rats weighing approximately 250–300 g were used for the experiment. All rats were maintained in a controlled temperature at 21–22 C, 45–55% humidity environment with a regular light and dark period (12 h light, 12 h dark), and fed with 24% protein added commercial rat diet ad libitum. The rats were randomly distributed into three groups: control (C) group (n: 7), procarbazine (P) treated group (n: 8), and procarbazine + melatonin (PM) treated group (n: 6, 2 rats from this group died during the experimental period). In the C group, the rats were fed with 0.5 mL serum physiologic (SF) peroral once a w similar with the other groups. In the P and PM treated groups, P was applied peroral by gavage once a w at a dose of 62.5 mg/kg/w for 4 w (total dose: 250 mg/kg). In the PM group, 10 mg/kg M was administered intraperitoneally daily at the same time (8 in the evening) in order to avoid circadian rhyme for 5 days per w for 4 w (total 20 d). The Experimentation Ethics Committee of our institution approved the experimental procedures of the study. Preparation of drugs Melatonin (Sigma Chemical, St Louis, MO) was administered intraperitoneally at a dose of 10 mg/kg dissolved in 0.5 ml saline under sterile conditions for 5 days per w for 4 weeks [Guven et al. 2011]. Procarbazine (NatulanÕ Sigma Tau, France) was administered peroral by gavage at a dose of 62.5 mg/kg/w for 4 w dissolved in 1 ml saline under sterile conditions once a w for 4 w [Parchuri et al. 1993]. Study design The rats were maintained for 90 d after the first dose of P as mentioned in the literature [Gocmen et al. 2001]. After the end of the study process, the rats were anesthetized with isofluorane gas anesthesia and blood removed by cardiac puncture into heparinized tubes for hormonal and antioxidant enzyme assays. The serum for determining antioxidant enzyme levels were placed into separate tubes and frozen in liquid nitrogen. The tissues were kept frozen at 80 C until assayed. The testis and epididymis were carefully dissected out, all fat and blood was trimmed. The right epididymis was removed and rinsed immediately in a plate that was filled with 2 ml of SF (for standardization of the ejaculate volume). After a 5 min incubation of the semen in SF, 50 microliters of this solution was added to a 450 microliter saline for a final concentration of 500 microliters for analyses. Fifty microliters from the final specimen was then placed into the sperm analyzer for analyzing. The left testis, epididymis, and seminal vesicle were removed and after washing in normal saline, their weight and volume were measured. After these measurements, the testis was placed into formaldehyde for histopathologic analyses.
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Sperm motility and morphology The motility and morphology was analyzed with the BiolaÕ automatic sperm analyzer model SFA-500 (Biola SFA-500, Russia) and the data was calculated with its original software Sa8 version 8.1194. The device is produced to analyze sperm of both humans and animals. The software was calibrated for use with rat sperm to analyze rats. For motility analyses, 50 microliters of the specimen was loaded into the analyzers special cuvette. Then the cuvette was placed into the analyzer for sperm counting. The analyzer automatically measured the sperm parameters including sperm concentration per ml and sperm motility (fast linear motility, slow linear motility).
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Biochemical analysis GPx activity measurement GPx activities in serum were measured by the method described previously [Sun et al. 1988]. The reaction mixture was 50 mmol/L tris buffer, pH 7.6 containing 1 mmol/L of Na2EDTA, 2 mmol/L of reduced glutathione (GSH), 0.2 mmol/L of NADPH, 4 mmol/L of sodium azide, and 1000 U of glutathione reductase (GR). Fifty ml of plasma and 950 ml of reaction mixture were mixed and incubated for 5 min at 37 C. Then the reaction was initiated with 8.8 mmol/L H2O2 and the decrease in NADPH absorbance was followed at 340 nm for 3 min. GPx activity was expressed in U/ml [Lawrence and Burk 2012]. CuZn-SOD activity measurement CuZn-SOD activity in serum was determined according to the method of Sun et al. [1988]. It is based on the inhibition of nitroblue tetrazolium (NBT) reduction by the xanthine/ xanthine oxidase system as a superoxide generator. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. CuZn-SOD activity was expressed in U/ml [Sun et al. 1988].
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After incubating for 10 min, the absorbance was measured spectrometrically at 540 nm. The nitrite/nitrate concentrations in the samples were calculated from a standard curve [Tracey et al. 1995]. Serum FSH, LH, and testosterone analyses used standard immunoassay methods. Serum follicle-stimulating hormone, LH, and testosterone levels were measured by ADVIA Centaur XP Immunoassay System using its kits (Siemens Healthcare Diagnostics Ltd., Erlangen, Germany). Histopathological analyses The testis specimens were fixed in 10% buffered formaldehyde. After routine pathological procedure, 5 micron section slides were prepared. Slides were stained with hematoxylen-eosine (HE). The slides were examined by a specialized pathologist blinded to all groups. Seminiferous tubules were counted and examined on a light microscope (40X, HPF). Forty-five seminiferous tubules were examined for each rat. Sertoli cells and spermatogonias were counted in each seminiferous tubule. Germinative layer and seminiferous tubule width and length were calculated. And then median levels of all 45 counts were calculated. Statistical analysis Results were expressed as the mean plus/minus standard error of the mean (mean ± S.E.M.). Numeric data were analyzed first using the Kruskal–Wallis test to determine differences between the groups, then the Mann–Whitney U-test was employed to analyze two groups consecutively. p values of 50.05 were regarded as significant. SPSS for windows program version 10.0 was used for the statistical analyses.
Declaration of interest The authors report no declarations of interest.
Author contributions MDA level measurement Serum MDA levels were determined by the method described previously. After the reaction of thiobarbituric acid with MDA, the reaction product was extracted in butanol and was measured spectrofluorometrically (excitation:532 nm, emission: 553 nm, slit 10 nm). After the reaction of thiobarbituric acid with MDA, the reaction product was measured spectrometrically. Tetramethoxy propane solution was used as standard. The MDA levels of serum were expressed as nmol/ml [Ohkawa et al. 1979]. NO 2 =NO3 level measurement Serum NO concentrations were determined by 2 =NO3 using the Griess reaction according to Tracey et al. [1995]. The reaction mixture consisted of reduced nicotineamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and nitrate reductase. After incubation of serum samples with reaction mixture, Griess reagent (a 1:1 mixture of 1% sulfanilamide in 5% H3PO4 and 0.1% N-[1-naphtyl]-ethylenediamine) was added to the samples.
All authors contributed extensively to the work presented in this paper. Conceived and designed the experiments: VK; Performed the experiments: VK, BFA, YP; Analyzed the data: VK, MG; Contributed reagents/materials/analysis tools: EOA, OE, NY; Pathologic examinations: NY; Wrote the manuscript: VK, BFA, MG, OB, EOA, NK, MS, NY; Revised the manuscript: VK, EM, BFA, MS. All authors approved the revisions to the manuscript.
References Atessahin, A., Sahna, E., Turk, G., Ceribasi, A.O., Yilmaz, S., Yuce, A., et al. (2006) Chemoprotective effect of melatonin against cisplatininduced testicular toxicity in rats. J Pineal Res 41:21–27. Gocmen, M., Akay, A.F., Sahin, H., Deniz, M., Nergiz, Y. and Bircan, M.K. (2001) The effects of GnRH analogues and antiandrogenes in preventing the gonadotoxic effects of COPP chemotherapy. Int Urol Nephrol 33:651–654. Guven, A., Uysal, B., Gundogdu, G., Oztas, E., Ozturk, H. and Korkmaz, A. (2011) Melatonin ameliorates necrotizing enterocolitis in a neonatal rat model. J Pediatr Surg 46:2101–2107. Horstman, M.G., Meadows, G.G. and Yost, G.S. (1987) Separate mechanisms for procarbazine spermatotoxicity and anticancer activity. Cancer Res 47:1547–1550.
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Notice of correction: The surname of the sixth author has been corrected since the original online publication date of August 20, 2014.
