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Beneficial effects of melatonin compared with allopurinol in experimental testicular torsion
Beneficial Effects of Melatonin Compared With Allopurinol in Experimental Testicular Torsion By Adnan Abasiyanik and Lu¨tfi Dag˘do¨nderen Konya, Turkey
Background/Purpose: Several antioxidant agents such as allopurinol have been used to prevent ischemia-reperfusion (I/R) injury–induced tissue damage after experimental testicular torsion so far. The current study was designed to determine the effect of melatonin, which is a potent antioxidant agent, in preventing testicular damage following torsion. Methods: Sixty prepubertal male Wistar-Albino rats were divided into 5 groups: control (C), torsion (T), torsion plus detorsion (TD), torsion plus allopurinol (200 mg/kg) plus detorsion (A), and torsion plus melatonin (50 mg/kg) plus detorsion (M). Left testes were rotated 720° for 6 hours. The torsed testes were detorsed. Detorsion time was 6 hours. In all groups, left orchiectomies were performed to determine the tissue levels of malondialdehyde (MDA) and histopathologic changes. Blood samples were taken to measure serum creatine phosphokinase (CPK) levels. The results were analyzed statistically.
MDA levels in group M were statistically different from groups T and TD (P ⬍ .05). However, in groups A and T, MDA levels were similar (P ⬎ .05). The highest histologic grade was determined in group TD (3.8 ⫾ 0.5). Histologic grade of group M was significantly lower than group TD (P ⬍ .001), but there was no histologic difference between testes of groups A and TD (P ⬎ .05). Conclusions: These results have shown that melatonin treatment prevents I/R injury both biochemically and histopathologically, whereas allopurinol treatment prevents it only biochemically in experimental testicular torsion. Melatonin is a potent antioxidant agent more effective than allopurinol in preventing testicular I/R injury. J Pediatr Surg 39:1238-1241. © 2004 Elsevier Inc. All rights reserved.
Results: Serum CPK levels of groups A and M were found to be significantly lower than groups T and TD (P ⬍ .05). Tissue
INDEX WORDS: Testicular torsion, melatonin, allopurinol, antioxidant agent.
T
phokinase (CPK) and its isoenzymes have a diagnostic value in tissue ischemia.8 Melatonin, which is secreted by the pineal gland, has been shown recently to be a potent free radical scavenger.9 It has both a lipophilic and hydrophilic compound.10 Thus, it freely permeates all morphophysiologic barriers of cells in any organ.11 This study was designed to investigate protective effect of melatonin, which is more effective than other free radical scavengers, in I/R injury after experimental testicular torsion.
REATMENT OF TESTICULAR torsion is immediate operative exploration of the scrotum. The severity of testicular damage is related to the time and degree of torsion.1 An aggressive surgical approach has improved testicular salvage rates.2 However, it is reported that there has been a testicular atrophy rate of 68% after testicular salvage.3 The main cause of testicular damage after torsion is oxygen free radicals produced during reperfusion.4 Oxygen free radicals oxidize membrane lipids, proteins, and DNA, leading to cellular dysfunction and sometimes cell death.5 They can produce mutations in the genetic code, inducing damage to DNA.6 Malondialdehyde (MDA) is produced during the attack of oxygen free radicals to membrane lipoproteins.7 MDA is used widely as an indicator of oxidative stress induced by ischemia-reperfusion (I/R). Creatine phosFrom the Department of Pediatric Surgery, Selc¸uk University, Medical Faculty, Konya, Turkey. Address reprint requests to Dr Adnan Abasiyanik, Aziziye Cad. Ko¨s¸em I˙s¸hanı No:2/102, 42030 Konya, Turkey. © 2004 Elsevier Inc. All rights reserved. 0022-3468/04/3908-0016$30.00/0 doi:10.1016/j.jpedsurg.2004.04.018 1238
MATERIALS AND METHODS
Animals and Experimental Procedures Sixty prepubertal male Wistar-Albino rats (120 to 180 g) were used in this study. Rats were obtained from the Selc¸uk University Experimental Research Center. They were housed in cages with 4 animals per cage and provided with standart rodent chow and water. All surgical procedures were performed under ketamine anesthesia (50 mg/kg intramuscularly) with sterile technique. Surgery was carried out through a left ilioinguinal incision. The left testis was rotated 720° in a clockwise direction and fixed within hemiscrotum with 4-0 polyglactin suture for 6 hours. The wound was closed with 4-0 silk suture. The torsed testes were detorsed. Detorsion time was 6 hours. In all groups, left orchiectomies were performed to determine the tissue levels of MDA and histopathologic changes, and 3-mL blood samples were taken to measure CPK levels. Journal of Pediatric Surgery, Vol 39, No 8 (August), 2004: pp 1238-1241
EFFECT OF MELATONIN ON TORSED TESTIS
1239
Table 1. Mean Values of Serum CPK and Tissue MDA Groups
CPK (IU/L)
MDA (nmol/g wet tissue)
C T TD A M
333.62 ⫾ 3.92*† 1195.20 ⫾ 108.21*† 1555.83 ⫾ 106.29*† 863.61 ⫾ 63.50† 746.46 ⫾ 61.14*
5.1 ⫾ 3.3*† 108.2 ⫾ 7.6† 161.9 ⫾ 15.8*† 100.6 ⫾ 35.7 77.9 ⫾ 22.7
NOTE. Values are expressed as mean ⫾ SD. *P ⬍ .05 compared with group A. †P ⬍ .05 compared with group M.
Animals were divided into 5 groups as follows. In group C (control group; n ⫽ 8) left orchiectomy was performed, and blood samples were taken to determine basal levels of biochemical parameters. In group T (torsion group; n ⫽ 10), left orchiectomy was performed, and blood samples were taken after 6-hour torsion. In group TD (torsion/detorsion group; n ⫽ 12), after 6-hour torsion and 6-hour detorsion, left orchiectomy was performed, and blood samples were taken. In group A (torsion/allopurinol treatment/detorsion group; n ⫽ 13), after 6-hour torsion, detorsion was performed. Allopurinol (Urokoliz, I˙lsan-iltas¸; 200 mg/kg) was injected intraperitoneally 15 minutes before detorsion. After 6-hour detorsion, left orchiectomy was performed, and blood samples were taken. In group M (torsion/melatonin treatment/detorsion group; n ⫽ 17), after 6-hour torsion, detorsion was performed. In this group, 50 mg/kg melatonin ([N-Acetyl-5-methoxytryptamine] M-250 Lot 25HO904, Sigma) was injected intraperitoneally 15 minutes before detorsion. After 6-hour detorsion, left orchiectomy was performed, and blood samples were taken.
Biochemical Analysis Blood samples were prepared for biochemical analysis, and serum CPK levels were measured in autoanalyser. Tissue MDA levels were determined as an index of lipid peroxidation as described by Uchiyama and Mihara.12 The MDA results were specified as nanomoles per gram of wet tissue.
Histopathologic Evaluation The specimens were fixed in Bouin’s solution for 24 hours and blocked in paraffin. The tissue samples were stained with H & E. All specimens were evaluated by a pathologist in a blinded fashion. Testicular tissue injury was graded on a system described by Cosentino et al.13 Grade 1 showed normal testicular architecture with an orderly arrangement of germinal cells. Grade 2 injury showed less orderly, noncohesive germinal cells and closely packed seminiferous tubules. Grade 3 injury exhibited disordered, sloughed germinal cells with shrunken, pyknotic nuclei and less distinct seminiferous tubule borders. Grade 4 injury defined seminiferous tubules that were packed closely with coagulative necrosis of the germinal cells.
In addition, there were statistically significant differences between CPK activities of groups M and A (P ⬍ .05). Tissue MDA Results MDA levels of group M were significantly lower when compared with groups T and TD (P ⬍ .05). However, in groups A and T, MDA levels were similar (P ⬎ .05). The mean MDA levels are shown in Table 1. Histopathologic Changes The highest histologic grade was determined in group TD (3.8 ⫾ 0.5). Most of the testes in group M showed grade 2 injury. Histologic grade of group M was significantly lower than group TD (P ⬍ .001). But there was no histologic difference between testes of groups A and TD (P ⬎ .05) Histopathologic evaluation is summarized in Table 2. DISCUSSION
Testicular torsion results in infertility and testicular damage. It has been shown that 1 hour of minimum time causes testicular damage after experimental testicular torsion in the rat.14 Thus, we formed a 6-hour testicular torsion model rotating left testis 720° to constitute a severe testicular damage. In recent years, several antioxidant agents have been used to prevent I/R-induced tissue damage in experimental testicular torsion such as superoxide dismutase (SOD), catalase, calcium channel blockers, oxypurinol, and allopurinol, except melatonin.15-17 There are several strategies to decrease the effect of oxygen free radicals after I/R. Allopurinol and oxypurinol inhibit the action of the enzyme xanthine oxidase.18 Melatonin, which is a potent antioxidant agent, not only is a direct scavenger of toxic hydroxyl radical, but it also stimulates the activity of the antioxidative enzyme glutathione peroxidase.19 Akhter et al15 used oxypurinol and polyethylene glycol-superoxide dismutase (PEG-SOD) to prevent reperfusion injury after 1 hour of testicular torsion. They found that these antioxidant agents prevent testicular damage histopathologically. Prillaman and Turner17 also studied to determine whether testicular function after 1 hour of torsion can be rescued by the administration of
Statistical Analysis Data were analyzed by 1-way analysis of variance (ANOVA) and Mann-Whitney U test. A level of P ⬍ .05 was accepted as statistically significant.
RESULTS
Serum CPK Results The mean CPK activities in all groups are shown in Table 1. CPK activities of groups M and A were found to be significantly lower than groups T and TD (P ⬍ .05).
Table 2. Histopathologic Evaluation of Groups Groups
Mean (⫾SD) Histologic Grade
Median Histologic Grade
C T TD A M
1.0 ⫾ 0.0*† 2.7 ⫾ 0.5 3.8 ⫾ 0.5† 3.0 ⫾ 0.7 2.4 ⫾ 0.7
1 3 4 3 2
*P ⬍ .05 compared with group A. †P ⬍ .05 compared with group M.
˘ DO ¨ NDEREN ABASIYANIK AND DAG
1240
antioxidant agents (SOD, catalase, verapamil, and allopurinol). These investigators showed that SOD and catalase treatments provide significant rescue of functioning testicular parenchyma after 1 hour of torsion, and neither allopurinol nor verapamil adds benefit. But the dose of allopurinol could have been inappropriate. No significant rescue was seen in testes undergoing 2 hours of torsion. However, Akgu¨ r et al16 reported that allopurinol treatment prevents reperfusion injury after testicular torsion lasting as long as 5 hours. They assessed thiobarbituric acid–reactive products of lipid peroxidation in testicular tissues. In this study, we indicated that allopurinol treatment decreases levels of CPK and MDA significantly (P ⬍ .05), but it does not sufficiently prevent testicular damage histopathologically after 6 hours of torsion (P ⬎ .05). Greenstein et al20 and Blank et al21 used antioxidant agents to prevent testicular damage after 3 hours and 6 hours of torsion, respectively. Similarly, they also found no palliative effects of antioxidant treatments (PEGSOD, diltiazem, respectively) on testicular damage histopathologically. However, in our study, we determined that melatonin treatment prevents I/R injury in testicular tissue both biochemically and histopathologically after 6 hours of torsion. These results have shown that antioxidant agents such as allopurinol, except melatonin, which is used to prevent testicular damage in experimental
torsion lasting more than 1 hour, prevent I/R injury biochemically but not histopathologically. We propose histopathologic evaluation made with biochemical analysis to determine the effect of antioxidant treatments to testicular tissue in testicular torsion. Melatonin scavenges high toxic peroxynitrite anion, which is a reaction product of superoxide and nitric oxide and induces oxidative damage.22 It is significantly more effective in physiologically tolerable concentrations when compared with free radical scavengers such as glutathione.23 Its lipophilicity and hydrophilicity ensure that melatonin rapidly enters cells where it may accumulate in the nucleus.24 Thus, melatonin protects DNA against oxidative damage.25,26 Kotler et al27 have shown that melatonin increases the mRNA levels for glutathione peroxidase and SOD in the rat brain cortex. In addition, melatonin modulates the androgen milieu and controls the immune response.28 Gorman and Yellon29 have shown that melatonin stimulates testis development as well. Melatonin is a potent antioxidant agent more effective than allopurinol in preventing testicular damage after acute experimental torsion. Even though this is an animal model, melatonin may clinically be used as an antioxidant agent in testicular torsion.
REFERENCES 1. Heindel RM, Pakyz RE, Reinking LN, et al: The effect of various degrees of unilateral spermatic cord torsion on fertility in the rat. J Urol 144:366-369, 1990 2. Anderson JB, Williams RC: Fertility after torsion of spermatic cord. Br J Urol 65:225-230, 1990 3. Krarup T: The testis after torsion. Br J Urol 50:43-46, 1978 4. Ambrosio G, Flaherty JT, Duilio C, et al: Oxygen radicals generated at reflow induce peroxidation of membrane lipids in reperfused hearts. J Clin Invest 87:2056-2066, 1991 5. Globus MY, Busto R, Lin B, et al: Detection of free radical activity during transient global ischemia and recirculation: Effects of intraischemic brain temperature modulation. J Neurochem 65:12501256, 1995 6. Ames BN: Endogenous oxidative DNA damage, aging, and cancer. Free Rad Res Commun 7:121-128, 1989 7. Floyd RA: Role of oxygen free radicals in carcinogenesis and brain ischemia. FASEB J 4:2587-2597, 1990 8. Erol D, Germiyanol˘u C, Bulut G, et al: Diagnostic value of creatine phosphokinase in testicular torsion. Int Urol Nephrol 24:201204, 1992 9. Pieri C, Marra M, Moroni F, et al: Melatonin: A peroxyl radical scavenger more potent than vitamin E. Life Sci 55:272-276, 1994 10. Shida CS, Castrucci AML, Lamy-Freund MT: High melatonin solubility in aqueous medium. J Pineal Res 16:198-201, 1994 11. Reiter RJ: Functional diversity of the pineal hormone melatonin: Its role as an antioxidant. Endocrinol Diabetes 104:10-16, 1996 12. Uchiyama M, Mihara M: Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 86:271-278, 1978 13. Cosentino MJ, Nishida M, Rabinowitz R, et al: Histopathology
of prepubertal rat testes subjected to various durations of spermatic cord torsions. J Androl 7:23-31, 1986 14. Turner TT, Brown KJ: Spermatic cord torsion: Loss of spermatogenesis despite return of blood flow. Biol Reprod 49:401-407, 1993 15. Akhter S, Sridher S, Katlowitz NM, et al: Immune response to testicular ischemia and reperfusion. J Urol 143:262 A, 1990 16. Akgu¨ r FM, Kilinc¸ K, Aktug˘ T, et al: The effect of allopurinol pretreatment before detorting testicular torsion. J Urol 151:1715-1717, 1994 17. Prillaman HM, Turner TT: Rescue of testicular function after acute experimental torsion. J Urol 157:340-345, 1997 18. Reilly PM, Schiller HJ, Bulkley GB: Pharmacologic approach to tissue injury mediated by free radical and other reactive oxygen metabolites. Am J Surg 161:488-503, 1991 19. Pablos MI, Chuang J, Reiter RJ, et al: Time course of the melatonin-induced increase in glutathione peroxidase activity in chick tissues. Biol Signals 4:325-330, 1995 20. Greenstein A, Smith-Harrison LI, Wakely PE, et al: The effect of polyethylene glycol-superoxide dismutase administration on histological damage following spermatic cord torsion. J Urol 148:639-641, 1992 21. Blank ML, O’Neill PJ, Steigman CK, et al: Reperfusion injury following testicular torsion and detorsion in prepubertal rats. Urol Res 21:389-393, 1993 22. Cuzzocrea S, Zingarelli B, Gilad E, et al: Protective effect of melatonin in carrageenan-induced models of local inflammation. J Pineal Res 23:106-116, 1997 23. Tan DX, Chen LD, Poeggeler B, et al: Melatonin: A potent, endogenous hydroxyl radical scavenger. Endocrine J 1:57-60, 1993 24. Menendez-Pelaez A, Poeggeler B, Reiter RJ, et al: Nuclear localization of melatonin in different mammalian tissues: Immunocy-
EFFECT OF MELATONIN ON TORSED TESTIS
totochemical and radioimmunoassay evidence. J Cell Biochem 53:373382, 1994 25. Cabrer J, Burkhardt S, Tan DX, et al: Autoxidation and toxicantinduced oxidation of lipid and DNA in monkey liver: Reduction of molecular damage by melatonin. Pharmacol Toxicol 89:225-230, 2001 26. Wakatsuki A, Okatani Y, Izumiya C, et al: Melatonin protects against ischemia and reperfusion-induced oxidative lipid and DNA damage in fetal rat brain. J Pineal Res 26:147-152, 1999 27. Kotler M, Rodriguez C, Sainz RM, et al: Melatonin increases
1241
gene expression for antioxidant enzymes in rat brain cortex. J Pineal Res 24:83-89, 1998 28. Valenti S, Giusti M: Melatonin participates in the control of testosterone secretion from rat testis: An overview of our experience. Ann N Y Acad Sci 966:284-289, 2002 29. Gorman MR, Yellon SM: Three daily melatonin infusions alter gonadal development but not GnRH neuron number in the medial preoptic area or diagonal band of Broca in Siberian hamsters. Neurosci Lett 210:165-168, 1996
Background/Purpose: Several antioxidant agents such as allopurinol have been used to prevent ischemia-reperfusion (I/R) injury–induced tissue damage after experimental testicular torsion so far. The current study was designed to determine the effect of melatonin, which is a potent antioxidant agent, in preventing testicular damage following torsion. Methods: Sixty prepubertal male Wistar-Albino rats were divided into 5 groups: control (C), torsion (T), torsion plus detorsion (TD), torsion plus allopurinol (200 mg/kg) plus detorsion (A), and torsion plus melatonin (50 mg/kg) plus detorsion (M). Left testes were rotated 720° for 6 hours. The torsed testes were detorsed. Detorsion time was 6 hours. In all groups, left orchiectomies were performed to determine the tissue levels of malondialdehyde (MDA) and histopathologic changes. Blood samples were taken to measure serum creatine phosphokinase (CPK) levels. The results were analyzed statistically.
MDA levels in group M were statistically different from groups T and TD (P ⬍ .05). However, in groups A and T, MDA levels were similar (P ⬎ .05). The highest histologic grade was determined in group TD (3.8 ⫾ 0.5). Histologic grade of group M was significantly lower than group TD (P ⬍ .001), but there was no histologic difference between testes of groups A and TD (P ⬎ .05). Conclusions: These results have shown that melatonin treatment prevents I/R injury both biochemically and histopathologically, whereas allopurinol treatment prevents it only biochemically in experimental testicular torsion. Melatonin is a potent antioxidant agent more effective than allopurinol in preventing testicular I/R injury. J Pediatr Surg 39:1238-1241. © 2004 Elsevier Inc. All rights reserved.
Results: Serum CPK levels of groups A and M were found to be significantly lower than groups T and TD (P ⬍ .05). Tissue
INDEX WORDS: Testicular torsion, melatonin, allopurinol, antioxidant agent.
T
phokinase (CPK) and its isoenzymes have a diagnostic value in tissue ischemia.8 Melatonin, which is secreted by the pineal gland, has been shown recently to be a potent free radical scavenger.9 It has both a lipophilic and hydrophilic compound.10 Thus, it freely permeates all morphophysiologic barriers of cells in any organ.11 This study was designed to investigate protective effect of melatonin, which is more effective than other free radical scavengers, in I/R injury after experimental testicular torsion.
REATMENT OF TESTICULAR torsion is immediate operative exploration of the scrotum. The severity of testicular damage is related to the time and degree of torsion.1 An aggressive surgical approach has improved testicular salvage rates.2 However, it is reported that there has been a testicular atrophy rate of 68% after testicular salvage.3 The main cause of testicular damage after torsion is oxygen free radicals produced during reperfusion.4 Oxygen free radicals oxidize membrane lipids, proteins, and DNA, leading to cellular dysfunction and sometimes cell death.5 They can produce mutations in the genetic code, inducing damage to DNA.6 Malondialdehyde (MDA) is produced during the attack of oxygen free radicals to membrane lipoproteins.7 MDA is used widely as an indicator of oxidative stress induced by ischemia-reperfusion (I/R). Creatine phosFrom the Department of Pediatric Surgery, Selc¸uk University, Medical Faculty, Konya, Turkey. Address reprint requests to Dr Adnan Abasiyanik, Aziziye Cad. Ko¨s¸em I˙s¸hanı No:2/102, 42030 Konya, Turkey. © 2004 Elsevier Inc. All rights reserved. 0022-3468/04/3908-0016$30.00/0 doi:10.1016/j.jpedsurg.2004.04.018 1238
MATERIALS AND METHODS
Animals and Experimental Procedures Sixty prepubertal male Wistar-Albino rats (120 to 180 g) were used in this study. Rats were obtained from the Selc¸uk University Experimental Research Center. They were housed in cages with 4 animals per cage and provided with standart rodent chow and water. All surgical procedures were performed under ketamine anesthesia (50 mg/kg intramuscularly) with sterile technique. Surgery was carried out through a left ilioinguinal incision. The left testis was rotated 720° in a clockwise direction and fixed within hemiscrotum with 4-0 polyglactin suture for 6 hours. The wound was closed with 4-0 silk suture. The torsed testes were detorsed. Detorsion time was 6 hours. In all groups, left orchiectomies were performed to determine the tissue levels of MDA and histopathologic changes, and 3-mL blood samples were taken to measure CPK levels. Journal of Pediatric Surgery, Vol 39, No 8 (August), 2004: pp 1238-1241
EFFECT OF MELATONIN ON TORSED TESTIS
1239
Table 1. Mean Values of Serum CPK and Tissue MDA Groups
CPK (IU/L)
MDA (nmol/g wet tissue)
C T TD A M
333.62 ⫾ 3.92*† 1195.20 ⫾ 108.21*† 1555.83 ⫾ 106.29*† 863.61 ⫾ 63.50† 746.46 ⫾ 61.14*
5.1 ⫾ 3.3*† 108.2 ⫾ 7.6† 161.9 ⫾ 15.8*† 100.6 ⫾ 35.7 77.9 ⫾ 22.7
NOTE. Values are expressed as mean ⫾ SD. *P ⬍ .05 compared with group A. †P ⬍ .05 compared with group M.
Animals were divided into 5 groups as follows. In group C (control group; n ⫽ 8) left orchiectomy was performed, and blood samples were taken to determine basal levels of biochemical parameters. In group T (torsion group; n ⫽ 10), left orchiectomy was performed, and blood samples were taken after 6-hour torsion. In group TD (torsion/detorsion group; n ⫽ 12), after 6-hour torsion and 6-hour detorsion, left orchiectomy was performed, and blood samples were taken. In group A (torsion/allopurinol treatment/detorsion group; n ⫽ 13), after 6-hour torsion, detorsion was performed. Allopurinol (Urokoliz, I˙lsan-iltas¸; 200 mg/kg) was injected intraperitoneally 15 minutes before detorsion. After 6-hour detorsion, left orchiectomy was performed, and blood samples were taken. In group M (torsion/melatonin treatment/detorsion group; n ⫽ 17), after 6-hour torsion, detorsion was performed. In this group, 50 mg/kg melatonin ([N-Acetyl-5-methoxytryptamine] M-250 Lot 25HO904, Sigma) was injected intraperitoneally 15 minutes before detorsion. After 6-hour detorsion, left orchiectomy was performed, and blood samples were taken.
Biochemical Analysis Blood samples were prepared for biochemical analysis, and serum CPK levels were measured in autoanalyser. Tissue MDA levels were determined as an index of lipid peroxidation as described by Uchiyama and Mihara.12 The MDA results were specified as nanomoles per gram of wet tissue.
Histopathologic Evaluation The specimens were fixed in Bouin’s solution for 24 hours and blocked in paraffin. The tissue samples were stained with H & E. All specimens were evaluated by a pathologist in a blinded fashion. Testicular tissue injury was graded on a system described by Cosentino et al.13 Grade 1 showed normal testicular architecture with an orderly arrangement of germinal cells. Grade 2 injury showed less orderly, noncohesive germinal cells and closely packed seminiferous tubules. Grade 3 injury exhibited disordered, sloughed germinal cells with shrunken, pyknotic nuclei and less distinct seminiferous tubule borders. Grade 4 injury defined seminiferous tubules that were packed closely with coagulative necrosis of the germinal cells.
In addition, there were statistically significant differences between CPK activities of groups M and A (P ⬍ .05). Tissue MDA Results MDA levels of group M were significantly lower when compared with groups T and TD (P ⬍ .05). However, in groups A and T, MDA levels were similar (P ⬎ .05). The mean MDA levels are shown in Table 1. Histopathologic Changes The highest histologic grade was determined in group TD (3.8 ⫾ 0.5). Most of the testes in group M showed grade 2 injury. Histologic grade of group M was significantly lower than group TD (P ⬍ .001). But there was no histologic difference between testes of groups A and TD (P ⬎ .05) Histopathologic evaluation is summarized in Table 2. DISCUSSION
Testicular torsion results in infertility and testicular damage. It has been shown that 1 hour of minimum time causes testicular damage after experimental testicular torsion in the rat.14 Thus, we formed a 6-hour testicular torsion model rotating left testis 720° to constitute a severe testicular damage. In recent years, several antioxidant agents have been used to prevent I/R-induced tissue damage in experimental testicular torsion such as superoxide dismutase (SOD), catalase, calcium channel blockers, oxypurinol, and allopurinol, except melatonin.15-17 There are several strategies to decrease the effect of oxygen free radicals after I/R. Allopurinol and oxypurinol inhibit the action of the enzyme xanthine oxidase.18 Melatonin, which is a potent antioxidant agent, not only is a direct scavenger of toxic hydroxyl radical, but it also stimulates the activity of the antioxidative enzyme glutathione peroxidase.19 Akhter et al15 used oxypurinol and polyethylene glycol-superoxide dismutase (PEG-SOD) to prevent reperfusion injury after 1 hour of testicular torsion. They found that these antioxidant agents prevent testicular damage histopathologically. Prillaman and Turner17 also studied to determine whether testicular function after 1 hour of torsion can be rescued by the administration of
Statistical Analysis Data were analyzed by 1-way analysis of variance (ANOVA) and Mann-Whitney U test. A level of P ⬍ .05 was accepted as statistically significant.
RESULTS
Serum CPK Results The mean CPK activities in all groups are shown in Table 1. CPK activities of groups M and A were found to be significantly lower than groups T and TD (P ⬍ .05).
Table 2. Histopathologic Evaluation of Groups Groups
Mean (⫾SD) Histologic Grade
Median Histologic Grade
C T TD A M
1.0 ⫾ 0.0*† 2.7 ⫾ 0.5 3.8 ⫾ 0.5† 3.0 ⫾ 0.7 2.4 ⫾ 0.7
1 3 4 3 2
*P ⬍ .05 compared with group A. †P ⬍ .05 compared with group M.
˘ DO ¨ NDEREN ABASIYANIK AND DAG
1240
antioxidant agents (SOD, catalase, verapamil, and allopurinol). These investigators showed that SOD and catalase treatments provide significant rescue of functioning testicular parenchyma after 1 hour of torsion, and neither allopurinol nor verapamil adds benefit. But the dose of allopurinol could have been inappropriate. No significant rescue was seen in testes undergoing 2 hours of torsion. However, Akgu¨ r et al16 reported that allopurinol treatment prevents reperfusion injury after testicular torsion lasting as long as 5 hours. They assessed thiobarbituric acid–reactive products of lipid peroxidation in testicular tissues. In this study, we indicated that allopurinol treatment decreases levels of CPK and MDA significantly (P ⬍ .05), but it does not sufficiently prevent testicular damage histopathologically after 6 hours of torsion (P ⬎ .05). Greenstein et al20 and Blank et al21 used antioxidant agents to prevent testicular damage after 3 hours and 6 hours of torsion, respectively. Similarly, they also found no palliative effects of antioxidant treatments (PEGSOD, diltiazem, respectively) on testicular damage histopathologically. However, in our study, we determined that melatonin treatment prevents I/R injury in testicular tissue both biochemically and histopathologically after 6 hours of torsion. These results have shown that antioxidant agents such as allopurinol, except melatonin, which is used to prevent testicular damage in experimental
torsion lasting more than 1 hour, prevent I/R injury biochemically but not histopathologically. We propose histopathologic evaluation made with biochemical analysis to determine the effect of antioxidant treatments to testicular tissue in testicular torsion. Melatonin scavenges high toxic peroxynitrite anion, which is a reaction product of superoxide and nitric oxide and induces oxidative damage.22 It is significantly more effective in physiologically tolerable concentrations when compared with free radical scavengers such as glutathione.23 Its lipophilicity and hydrophilicity ensure that melatonin rapidly enters cells where it may accumulate in the nucleus.24 Thus, melatonin protects DNA against oxidative damage.25,26 Kotler et al27 have shown that melatonin increases the mRNA levels for glutathione peroxidase and SOD in the rat brain cortex. In addition, melatonin modulates the androgen milieu and controls the immune response.28 Gorman and Yellon29 have shown that melatonin stimulates testis development as well. Melatonin is a potent antioxidant agent more effective than allopurinol in preventing testicular damage after acute experimental torsion. Even though this is an animal model, melatonin may clinically be used as an antioxidant agent in testicular torsion.
REFERENCES 1. Heindel RM, Pakyz RE, Reinking LN, et al: The effect of various degrees of unilateral spermatic cord torsion on fertility in the rat. J Urol 144:366-369, 1990 2. Anderson JB, Williams RC: Fertility after torsion of spermatic cord. Br J Urol 65:225-230, 1990 3. Krarup T: The testis after torsion. Br J Urol 50:43-46, 1978 4. Ambrosio G, Flaherty JT, Duilio C, et al: Oxygen radicals generated at reflow induce peroxidation of membrane lipids in reperfused hearts. J Clin Invest 87:2056-2066, 1991 5. Globus MY, Busto R, Lin B, et al: Detection of free radical activity during transient global ischemia and recirculation: Effects of intraischemic brain temperature modulation. J Neurochem 65:12501256, 1995 6. Ames BN: Endogenous oxidative DNA damage, aging, and cancer. Free Rad Res Commun 7:121-128, 1989 7. Floyd RA: Role of oxygen free radicals in carcinogenesis and brain ischemia. FASEB J 4:2587-2597, 1990 8. Erol D, Germiyanol˘u C, Bulut G, et al: Diagnostic value of creatine phosphokinase in testicular torsion. Int Urol Nephrol 24:201204, 1992 9. Pieri C, Marra M, Moroni F, et al: Melatonin: A peroxyl radical scavenger more potent than vitamin E. Life Sci 55:272-276, 1994 10. Shida CS, Castrucci AML, Lamy-Freund MT: High melatonin solubility in aqueous medium. J Pineal Res 16:198-201, 1994 11. Reiter RJ: Functional diversity of the pineal hormone melatonin: Its role as an antioxidant. Endocrinol Diabetes 104:10-16, 1996 12. Uchiyama M, Mihara M: Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 86:271-278, 1978 13. Cosentino MJ, Nishida M, Rabinowitz R, et al: Histopathology
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