- Email: [email protected]
The effect of caffeic acid phenethyl ester (CAPE) on histopathological changes in testicular ischemia-reperfusion injury
Pharmacological Research 54 (2006) 293–297
The effect of caffeic acid phenethyl ester (CAPE) on histopathological changes in testicular ischemia-reperfusion injury Esin Atik a,∗ , Sadık G¨or¨ur b , Ahmet Namık Kiper b a
Department of Pathology, Mustafa Kemal University, Faculty of Medicine, Antakya, Hatay, Turkey Department of Urology, Mustafa Kemal University, Faculty of Medicine, Antakya, Hatay, Turkey
b
Accepted 13 June 2006
Abstract Testicular torsion causes an enhanced formation of reactive oxygen species which contributes to the pathophysiology of ischemia-reperfusion injury in the testis. We evaluated here the effect of caffeic acid phenethyl ester (CAPE), a new antioxidant and anti-inflammatory agent on histopathological changes in testicular ischemia-reperfusion injury. Adult male Wistar rats were divided into six groups of five each: control group 1 (n = 5), sham operation group 2 (n = 5), torsion/detorsion (T/D) group 3 (n = 5), T/D + saline group 4 (n = 5), T/D + CAPE group 5 (n = 5) and T/D + CAPE group 6 (n = 5). Group 1 served to determine baseline values of histopathological parameters, group 2 animals that underwent sham operation served as a control, while groups 3–6 animals were subjected to left unilateral torsion (2 h) and detorsion (24 h) periods. All the groups were sacrified 24 h later except group 6. CAPE was injected 2 days with the same dose to the group 6 and it was sacrified 48 h later. One testis removed and fixed in Bouin’s solution. After routine tissue processing myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) immunohistochemical methods were studied from paraffin embedded tissues. Treating rats with CAPE (applied at 10 mol/kg, 30 min prior to T/D) attenuated the testicular injury and as well as the tissue levels of MPO. At the same time testis tissue showed a decrease in iNOS activity. Our results suggest that CAPE treatment have a protective role on testicular T/D and this effect may be due to inhibiting the neutrophil mediated cellular injury. © 2006 Elsevier Ltd. All rights reserved. Keywords: Testicular ischemia-reperfusion; Nitric oxide; Myeloperoxidase; Caffeic acid phenethyl ester
1. Introduction Testicular torsion is the most common genital trauma of the adolescent boy and has been implicated in testicular injury, altered hormone production, subfertility and infertility [1–3]. The testicular injury is related with the duration and degree of torsion [1–3]. Testicular artery occlusion causes an enhanced formation of reactive oxygen species, which contributes to the pathophysiology of tissue damage [1]. After torsion and reoxygenation, the imbalance between restoration of oxygen sup-
Abbreviations: MPO, myeloperoxidase; iNOS, inducible nitric oxide synthases; NO, nitric oxide; NOS, nitric oxide synthase; CAPE, caffeic acid phenethyl ester; T/D, torsion/detorsion; ROS, reactive oxygen species; I/R, ischemia/reperfusion ∗ Corresponding author. Tel.: +90 326 214 06 49x142/532 735 89 03; fax: +90 326 214 49 77. E-mail address: [email protected] (E. Atik). 1043-6618/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2006.06.005
ply and mitochondrial respiratory function results in the massive generation of superoxide in mitochondria [1]. Previous studies in the rat have demonstrated that ischemiainducing testicular torsion followed by torsion repair and reperfusion resulted in neutrophil adhesion to subtunical venules, an increase in reactive oxygen species and increased expression of several apoptosis-associated molecules [4]. Many laboratories previously showed biochemical changes about this but there is not any detailed immunohistochemical study. Nitric oxide (NO), a freely diffusible, water-and-lipid soluble gaseous molecule with a short half-life is formed from l-arginine and molecular oxygen by a family of nitric oxide synthases (NOS) [5–8]. In recent studies NO has been implicated as a mediator of the inflammatory cytokines at the hypothalamicpituitary level [9]. Expression of iNOS requires transcriptional activation that is induced by various cytokines. The up-regulation of iNOS leads to excessive NO production for prolonged periods of
294
E. Atik et al. / Pharmacological Research 54 (2006) 293–297
time and accounts for oxyradical-mediated damage in several organs [5–8,10]. NO is an important mediator of cell death either through apoptosis or necrosis, depending on the intensity and the duration of injury [5–8,10]. Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis extracts and has been used as a folk medicine for many years. CAPE was known to have antioxidant, anti-inflammatory, antimicrobial, immunomodulatory and antineoblastic effects [1,8,11]. It has been shown to inhibit lipooxygenase activities as well as suppress lipid peroxidation. In addition CAPE inhibits the growth of different types of transformed cells and inhibits phorbol ester-induced hydrogen peroxide production and tumour promotion [8,12]. At a concentration of 10 mol CAPE completely blocks the production of ROS in human neutrophils [13]. The balance between the production and catabolism oxidants by cells and tissues is critical for maintenance of the biological integrity of the tissues [8,12]. We aimed in this study to evaluate the effect of caffeic acid phenethyl ester (CAPE), a new antioxidant and antiinflammatory agent on histopathological changes in testicular ischemia-reperfusion injury. 2. Materials and methods Thirty adult male Wistar rats weighing between 210 and 250 g were divided into six groups. They were acclimatized for 1 week before experimentation. The animals fed with a standard diet, kept on a physiological day–night rhythm and maintained in 24 ◦ C during the experimental procedures. The ethical committee on animal research at our institution approved the protocol for all animal experiments. We provided appropriate care and use of the laboratory animals as recommended by the Board of Registry publication guidelines. The CAPE was synthesized by the standard method of Grunberger et al. [14] and administered intraperitoneally once a day at a dose of 10 mol/kg body weight. Adult male Wistar rats were divided into six groups of five each: control group 1 (n = 5), sham operation group 2 (n = 5), torsion/detorsion (T/D) group 3 (n = 5), T/D + saline group 4 (n = 5), T/D + CAPE group 5 (n = 5) and T/D + CAPE group 6 (n = 5). Group 1: One group served to determine baseline values of histopathological parameters. Group 2: Group 2 that underwent sham operation served as an additional control (in this group a sham procedure was performed). Rats were anesthetized with ketamine (30 mg/kg body weight intraperitoneally). The testis was rotated 720◦ clockwise, it was immediately relieved and a 4-0 silk suture was placed through the tunica albuginea. Group 3: In the torsion–detorsion (T/D) group torsion was created by rotating the left testis 720◦ clockwise and maintained by placed through the tunica albuginea. After 2 h of torsion the testis was counter rotated back to the natural position and reinserted into the scrotum. Group 4: In this group the same surgical procedure was done as in the T/D group but saline was injected intraperitoneally at a dose of 2 cc before detorsion and closure.
Groups 5 and 6: In the groups receiving CAPE before detorsion the same surgical procedure was done as in the T/D group but CAPE was injected intraperitoneally at a dose of 10 mol/kg body weight before detorsion and closure. Others were subjected to left unilateral torsion (2 h) and detorsion (24 h) periods. All the groups were sacrified 24 h later except group 6. CAPE was injected 2 days with the same dose to the group 6 and it was sacrified 48 h later. This is an extra planned group just to detect iNOS and MPO values after 48 h. So it was compared with especially group 5 and other groups. The rats were sacrified by pentobarbital overdose after unilateral orchiectomy was performed on the rats. Testis tissues were put into Bouin’s solution. After routine tissue processing myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) immunohistochemical methods were studied from paraffin embedded tissues. Immunohistochemical staining was carried out by deparaffinization, dehydradation and incubation in citrate buffer. Single antigen staining was performed with iNOS protein, and Ultra Vision Polyvalent, Rabbit, HRP-AEC kit (Neomarkers-Biogen, Lab Vision Corp., USA). Another antigen staining was performed with MPO protein, Ultra Vision Polyvalent, Rabbit, HRP-AEC kit (Neomarkers-Biogen, Lab Vision Corp., USA). The number of cytoplasmic iNOS and MPO positive stained cells per field was counted by evaluation of atleast dense stained five fields, at a magnification of 40× by an Olympus BX51 light microscope. iNOS is expressed constitutively in leydig cells, sertoli cells and spermatogenic cells in the normal rat testis [9]. Because of that iNOS positive stained germ cells were excepted in control group 1. The statistical procedures carried out by the software of Epi INFO version 3.3 and P < 0.05 was considered statistically significant. For the comparison of the findings Kruskall–Wallis and Spearman’s correlation tests were performed. 3. Results Results were shown in Table 1. The target cell population of MPO and iNOS immunostaining is neutrophil leucocytes and mononuclear cells, respectively. The iNOS expressions in the control group were in baseline values (Fig. 1). Ischemia and reperfusion caused to increase the expression of testicular iNOS in this study (Fig. 2). In the sham operation group the mean value of iNOS immunoexpression was 26.0 ± 9.6 and the mean value of MPO immunoexpression was 38.0 ± 8.3. These values were significantly higher in T/D and T/D + saline groups. The mean immunostaining values of iNOS were 90.0 ± 17 and 95.0 ± 29.5 in T/D and T/D + saline groups, respectively. The mean immunoexpression values of MPO were179.6 ± 37.8 and 179.0 ± 38.4 in the same groups, respectively. It was demonstrated that histopathologically ischemia and reperfusion increases the expression of testicular MPO also in this study (Fig. 3). Whereas there was a significant decrease of iNOS (Fig. 4) and MPO levels in the CAPE groups. The differences of iNOS and MPO values between groups 5 and 6 were not statistically significant. But the iNOS and MPO values of in these groups were lower
E. Atik et al. / Pharmacological Research 54 (2006) 293–297
295
Table 1 Descriptive statistics of iNOS and MPO by the intervention groups (Kruskall–Wallis test) Statistical values
Intervention groups
iNOS (χ2 = 25.9, P < 0.001)
Control Sham Torsion/detorsion Torsion/detorsion + saline Torsion/detorsion + CAPE 24 h Torsion/detorsion + CAPE 48 h
n
Total
MPO (χ2 = 26.9, P < 0.001)
Control Sham Torsion/detorsion Torsion/detorsion + saline Torsion/detorsion + CAPE 24 h Torsion/detorsion + CAPE 48 h
Total
Mean
S.D.
5 5 5 5 5 5
0 26.0 90.0 95.0 66.0 62.0
0 9.6 17.0 29.5 6.5 9.0
30
56.5
37.0
5 5 5 5 5 5
0 38.0 179.6 179.0 80.0 84.0
0 8.3 37.8 38.4 11.7 17.1
30
93.4
71.3
n, the numbers of animals in each group.
Figs. 1 and 2. Fig. 1. iNOS positive stained constitutional cells in a normal testis from control group (iNOS × 400). Fig. 2. The increased amount of iNOS positive stained cells in T/D group (iNOS × 400).
Fig. 5. (A) T/D group displays a dense inflammatory infiltrate, oedema, necrosis and congestion (hematoxylin–eosine × 200). (B) The testis with normal histopathologic appearance in CAPE administrated group (hematoxylin–eosine × 400).
than groups 3 and 4. A dense inflammatory infiltrate, oedema and congestion were seen in TD group by hematoxylin–eosine staining (Fig. 5A). CAPE administration caused a histopathologically normal appearance in testis (Fig. 5B). There was a positive correlation between iNOS and MPO levels (r = 0.911 and P < 0.001 Spearman’s correlation). 4. Discussion
Figs. 3 and 4. Fig. 3. The collections of inflammatory cells positive stained by MPO in T/D group (MPO × 200). Fig. 4. Decreased number of MPO positive stained cells in CAPE administrated group (MPO × 200).
Testicular torsion is a surgical emergency. Late presentation or failure to diagnose this condition leads to testicular injury and subfertility [10,15]. Testicular injury due to torsion and detorsion is an ischemia-reperfusion (IR) injury attributed to neutrophil infiltration and generation of reactive oxygen species (ROS). In the present study we detected significantly increased iNOS and MPO values in T/D and T/D + saline groups. Current studies revealed that testicular torsion–detorsion induces histological and biochemical changes as a result of ischemia-reperfusion injury [8,10,15]. Although reperfusion is
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E. Atik et al. / Pharmacological Research 54 (2006) 293–297
essential for the survival of ischemic tissue, the main pathophysiology is caused by the twisted spermatic cord and its release [8,10,15]. We demonstrated that histopathologically ischemia and reperfusion increases the expressions of testicular iNOS and MPO in this study. These high levels were compared to the control and sham groups suggest that iNOS and MPO activities may increase under abnormal conditions such as hypooxygenation. MPO is a major neutrophil protein. It is stored in azurophilic granules and released during phagocytosis [1]. MPO derived oxidants damage cells and tissue in vitro [1]. Ischemia leads to the activation of leukocytes trapped in the testicular vasculature, which may result in the release of chemotactic factors during reperfusion. Such activated neutrophils produce oxygen free radicals. These toxic oxygen derivatives cause a destructive effect on the adjacent tissue [1]. Lysiak et al. have also noted germ cell specific apoptosis after testicular IR and that an influx of neutrophils to the testis was essential for this pathology [16]. This germ cell specific apoptosis leads to a decrease in testicular weight and the loss of spermatogenesis [16]. NO is formed from l-arginine and molecular oxygen by nitric oxide synthases. Expression of iNOS requires various cytokines. The up-regulation of iNOS leads to excessive NO production and accounts for oxyradical-mediated damage in several organs. NO is an important mediator of cell death either through apoptosis or necrosis [5]. Otherwise sustained production of NO by iNOS in response to inflammatory stimuli has been shown to mediate antimicrobial and antitumoral activities [7]. Paradoxically oxygen is both essential for life as well as, toxic to human. It has been demonstrated that free oxygen radicals causing injury in post-ischemic cells and tissue with a critical mechanism [11]. Testicular injury due to T/D is an ischemia-reperfusion injury attributed to neutrophil infiltration, generation of ROS and reactive nitrogen species. ROS and reactive nitrogen species such as nitric oxide cause DNA damage. DNA damage can lead to loss of cell viability [3,10,11,15–17]. Ischemia causes an increase in NOS activity in the vascular endothelium and, later in a range of cells including infiltrating neutrophils and macrophages. While NO levels increase during ischemia, reperfusion leads to the generation of the superoxide radicals through the action of several mechanisms; the interaction between NO and O2 produces peroxynitrite. The breakdown of NO by O2 anions may be a factor in the decreased NO levels [8,17]. We found that increased expression of iNOS in T/D and T/D + saline groups, which is consistent with literature [17]. However, NO levels were a little increased in ischemia but decreased in reperfusion according to studies of Uz et al. [1] and Koltuksuz et al. [8]. Es¸refo˘glu et al. have demonstrated that the role of oxidative injury on testicular damage following myocardial IR injury and the increase of NO. They also noted inhibition of NO by CAPE and prevented testicular damage [17]. Despite intensive research, the role of NO remains uncertain in post-ischemic tissue injury and NO production is increased or decreased?
In another study XO was reported to catalyze the reduction of NO3 and NO2 to NO under hypoxic conditions [8]. We also demonstrated that CAPE administration brought the iNOS and MPO immunoexpressions around the sham values. CAPE administration attenuates the increase in the tissue MPO and iNOS levels. As a result we can say, – the beneficial effect of CAPE on testicular damage after T/D may be depend on CAPE’s antineutrophil effects – prophylactic CAPE administration in testicular ischemia may prevent reperfusion injury by eliminating reactive nitrogen species. We demonstrated that T/D increases the immunoexpression values of testicular iNOS and MPO. The administration of CAPE prevents the increase in immunoexpression values of iNOS and MPO, thus CAPE may be useful in dealing with ischemiareperfusion injuries. These results partially explain the pathophysiology of testis injury after IR and suggest that inhibition of iNOS and MPO by CAPE may be a novel pharmacological approach to prevent cell injury. However, further studies are required to clarify the exact mechanism of IR injury and effects of CAPE. Acknowledgement We are very grateful to Dr. Tacettin ˙Inandı for his kindly helping about statistical analyse. References ¨ [1] Uz E, S¨og˘ u¨ t S, S¸ahin S¸, Var A, Ozyurt H, G¨ulec¸ M, et al. The protective role of caffeic acid phenethyl ester (CAPE) on testicular tissue after testicular torsion and detorsion. World J Urol 2002;20:264–70. [2] Romeo C, Antonuccio P, Esposito M, Marini H, Impellizzeri P, Turiaco N, et al. Raxofelast, a hydrophilic vitamin e-like antioxidant, reduces testicular ischemia-reperfusion injury. Urol Res 2004;32:367–71. [3] Uguralp S, Karabulut AB, Mızrak B, Kaymaz F, Kızıltay F, Hasirci N. The effect of sustained and local administration of epidermal growth factor on improving bilateral testicular tissue after torsion. Urol Res 2004;32:323–31. [4] Lysiak JJ, Turner SD, Nguyen QA, Singbartl K, Ley K, Turner TT. Essential role of neutrophils in germ cell-specific apoptosis following ischemia/reperfusion injury of the mouse testis. Biol Reprod 2001;65:718–25. [5] Shiraishi K, Naito K, Yoshida Ki. Nitric oxide promotes germ cell necrosis in the delayed phase after experimental testicular torsion of rat. Biol Reprod 2001;65:514–21. [6] Fujisawa M, Tatsumi N, Fujioka H, Kanzaki M, Okuda Y, Arakawa S. Nitric oxide production of rat Leydig and Sertoli cells is stimulated by round spermatic factors. Mol Cell Endocrinol 2000;160:99–105. [7] Bauche F, Stephan JP, Touzalin AM, Jegou B. In vitro regulation of an inducible-type NO synthase in the rat seminiferous tubule cells. Biol Reprod 1998;58:431–8. ¨ [8] Koltuksuz U, Irmak MK, Karaman A, Uz E, Var A, Ozyurt H, et al. Testicular nitric oxide levels after unilateral testicular torsion/detorsion in rats pretreated with caffeic acid phenethyl ester. Urol Res 2000;28:360–3. [9] O’Bryan MK, Schlatt S, Gerdprasert O, Phillips DJ, Kretser DM, Hedger MP. Inducible nitric oxide synthase in the rat testis: evidence for potential roles in both normal function and inflammation-mediated infertility. Biol Reprod 2000;63:1285–93. [10] Bozlu M, Eskandari G, C¸ayan S, Canpolat B, Akbay E, Atik U, et al. The effect of poly(adenosine diphosphate-ribose) polymerase inhibitors
E. Atik et al. / Pharmacological Research 54 (2006) 293–297 on biochemical changes in testicular ischemia-reperfusion injury. J Urol 2003;169:1870–3. ¨ T¨urk¨oz Y, Hasc¸alık S¸, Hasc¸alık M, Cigremis Y, Mızrak B, et [11] C¸elik O, al. The protective effect of caffeic acid phenethyl ester on ischemiareperfusion injury in rat ovary. Eur J Obstet Gyn R B 2004;117(2): 183–8. ¨ ¨ Iraz M, S¨og˘ u¨ t S, Ozu˘ ¨ gurlu F, Ozyurt ¨ [12] Ozen S, Akyol O, F, et al. Role of caffeic acid phenethyl ester, an active component of propolis, against cisplatin-induced nephrotoxicity in rats. J Appl Toxicol 2004;24: 27–35. [13] Sud’ina GF, Mirzoeva OK, Pushkareva MA, Korshunova GA, Sumbatyan NV, Varfolomeev SD. Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett 1993;329:21–4.
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[14] Grunberger D, Banerjee R, Eisinger K, Oltz EM, Efros L, Caldwell M, et al. Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis. Experientia 1988;44:230–2. [15] Bozlu M, Cos¸kun B, C¸ayan S, Acar D, Aktas¸ S, Ulusoy E, et al. Inhibition of poly(adenosine diphosphate-ribose) polymerase decreases long-term histologic damage in testicular ischemia-reperfusion injury. Urology 2004;63:791–5. [16] Lysiak JJ, Nguyen QAT, Turner TT. Peptide and non-peptide reactive oxygen scavengers provide partial rescue of the testis after torsion. J Androl 2002;23:400–9. [17] Es¸refo˘glu M, G¨ul M, Parlakpınar H, Acet A. Effects of melatonin and caffeic acid phenethyl ester on testicular injury induced by myocardial ischemia/reperfusion in rats. Fundam Clin Pharmacol 2005;19:365–72.
The effect of caffeic acid phenethyl ester (CAPE) on histopathological changes in testicular ischemia-reperfusion injury Esin Atik a,∗ , Sadık G¨or¨ur b , Ahmet Namık Kiper b a
Department of Pathology, Mustafa Kemal University, Faculty of Medicine, Antakya, Hatay, Turkey Department of Urology, Mustafa Kemal University, Faculty of Medicine, Antakya, Hatay, Turkey
b
Accepted 13 June 2006
Abstract Testicular torsion causes an enhanced formation of reactive oxygen species which contributes to the pathophysiology of ischemia-reperfusion injury in the testis. We evaluated here the effect of caffeic acid phenethyl ester (CAPE), a new antioxidant and anti-inflammatory agent on histopathological changes in testicular ischemia-reperfusion injury. Adult male Wistar rats were divided into six groups of five each: control group 1 (n = 5), sham operation group 2 (n = 5), torsion/detorsion (T/D) group 3 (n = 5), T/D + saline group 4 (n = 5), T/D + CAPE group 5 (n = 5) and T/D + CAPE group 6 (n = 5). Group 1 served to determine baseline values of histopathological parameters, group 2 animals that underwent sham operation served as a control, while groups 3–6 animals were subjected to left unilateral torsion (2 h) and detorsion (24 h) periods. All the groups were sacrified 24 h later except group 6. CAPE was injected 2 days with the same dose to the group 6 and it was sacrified 48 h later. One testis removed and fixed in Bouin’s solution. After routine tissue processing myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) immunohistochemical methods were studied from paraffin embedded tissues. Treating rats with CAPE (applied at 10 mol/kg, 30 min prior to T/D) attenuated the testicular injury and as well as the tissue levels of MPO. At the same time testis tissue showed a decrease in iNOS activity. Our results suggest that CAPE treatment have a protective role on testicular T/D and this effect may be due to inhibiting the neutrophil mediated cellular injury. © 2006 Elsevier Ltd. All rights reserved. Keywords: Testicular ischemia-reperfusion; Nitric oxide; Myeloperoxidase; Caffeic acid phenethyl ester
1. Introduction Testicular torsion is the most common genital trauma of the adolescent boy and has been implicated in testicular injury, altered hormone production, subfertility and infertility [1–3]. The testicular injury is related with the duration and degree of torsion [1–3]. Testicular artery occlusion causes an enhanced formation of reactive oxygen species, which contributes to the pathophysiology of tissue damage [1]. After torsion and reoxygenation, the imbalance between restoration of oxygen sup-
Abbreviations: MPO, myeloperoxidase; iNOS, inducible nitric oxide synthases; NO, nitric oxide; NOS, nitric oxide synthase; CAPE, caffeic acid phenethyl ester; T/D, torsion/detorsion; ROS, reactive oxygen species; I/R, ischemia/reperfusion ∗ Corresponding author. Tel.: +90 326 214 06 49x142/532 735 89 03; fax: +90 326 214 49 77. E-mail address: [email protected] (E. Atik). 1043-6618/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2006.06.005
ply and mitochondrial respiratory function results in the massive generation of superoxide in mitochondria [1]. Previous studies in the rat have demonstrated that ischemiainducing testicular torsion followed by torsion repair and reperfusion resulted in neutrophil adhesion to subtunical venules, an increase in reactive oxygen species and increased expression of several apoptosis-associated molecules [4]. Many laboratories previously showed biochemical changes about this but there is not any detailed immunohistochemical study. Nitric oxide (NO), a freely diffusible, water-and-lipid soluble gaseous molecule with a short half-life is formed from l-arginine and molecular oxygen by a family of nitric oxide synthases (NOS) [5–8]. In recent studies NO has been implicated as a mediator of the inflammatory cytokines at the hypothalamicpituitary level [9]. Expression of iNOS requires transcriptional activation that is induced by various cytokines. The up-regulation of iNOS leads to excessive NO production for prolonged periods of
294
E. Atik et al. / Pharmacological Research 54 (2006) 293–297
time and accounts for oxyradical-mediated damage in several organs [5–8,10]. NO is an important mediator of cell death either through apoptosis or necrosis, depending on the intensity and the duration of injury [5–8,10]. Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis extracts and has been used as a folk medicine for many years. CAPE was known to have antioxidant, anti-inflammatory, antimicrobial, immunomodulatory and antineoblastic effects [1,8,11]. It has been shown to inhibit lipooxygenase activities as well as suppress lipid peroxidation. In addition CAPE inhibits the growth of different types of transformed cells and inhibits phorbol ester-induced hydrogen peroxide production and tumour promotion [8,12]. At a concentration of 10 mol CAPE completely blocks the production of ROS in human neutrophils [13]. The balance between the production and catabolism oxidants by cells and tissues is critical for maintenance of the biological integrity of the tissues [8,12]. We aimed in this study to evaluate the effect of caffeic acid phenethyl ester (CAPE), a new antioxidant and antiinflammatory agent on histopathological changes in testicular ischemia-reperfusion injury. 2. Materials and methods Thirty adult male Wistar rats weighing between 210 and 250 g were divided into six groups. They were acclimatized for 1 week before experimentation. The animals fed with a standard diet, kept on a physiological day–night rhythm and maintained in 24 ◦ C during the experimental procedures. The ethical committee on animal research at our institution approved the protocol for all animal experiments. We provided appropriate care and use of the laboratory animals as recommended by the Board of Registry publication guidelines. The CAPE was synthesized by the standard method of Grunberger et al. [14] and administered intraperitoneally once a day at a dose of 10 mol/kg body weight. Adult male Wistar rats were divided into six groups of five each: control group 1 (n = 5), sham operation group 2 (n = 5), torsion/detorsion (T/D) group 3 (n = 5), T/D + saline group 4 (n = 5), T/D + CAPE group 5 (n = 5) and T/D + CAPE group 6 (n = 5). Group 1: One group served to determine baseline values of histopathological parameters. Group 2: Group 2 that underwent sham operation served as an additional control (in this group a sham procedure was performed). Rats were anesthetized with ketamine (30 mg/kg body weight intraperitoneally). The testis was rotated 720◦ clockwise, it was immediately relieved and a 4-0 silk suture was placed through the tunica albuginea. Group 3: In the torsion–detorsion (T/D) group torsion was created by rotating the left testis 720◦ clockwise and maintained by placed through the tunica albuginea. After 2 h of torsion the testis was counter rotated back to the natural position and reinserted into the scrotum. Group 4: In this group the same surgical procedure was done as in the T/D group but saline was injected intraperitoneally at a dose of 2 cc before detorsion and closure.
Groups 5 and 6: In the groups receiving CAPE before detorsion the same surgical procedure was done as in the T/D group but CAPE was injected intraperitoneally at a dose of 10 mol/kg body weight before detorsion and closure. Others were subjected to left unilateral torsion (2 h) and detorsion (24 h) periods. All the groups were sacrified 24 h later except group 6. CAPE was injected 2 days with the same dose to the group 6 and it was sacrified 48 h later. This is an extra planned group just to detect iNOS and MPO values after 48 h. So it was compared with especially group 5 and other groups. The rats were sacrified by pentobarbital overdose after unilateral orchiectomy was performed on the rats. Testis tissues were put into Bouin’s solution. After routine tissue processing myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) immunohistochemical methods were studied from paraffin embedded tissues. Immunohistochemical staining was carried out by deparaffinization, dehydradation and incubation in citrate buffer. Single antigen staining was performed with iNOS protein, and Ultra Vision Polyvalent, Rabbit, HRP-AEC kit (Neomarkers-Biogen, Lab Vision Corp., USA). Another antigen staining was performed with MPO protein, Ultra Vision Polyvalent, Rabbit, HRP-AEC kit (Neomarkers-Biogen, Lab Vision Corp., USA). The number of cytoplasmic iNOS and MPO positive stained cells per field was counted by evaluation of atleast dense stained five fields, at a magnification of 40× by an Olympus BX51 light microscope. iNOS is expressed constitutively in leydig cells, sertoli cells and spermatogenic cells in the normal rat testis [9]. Because of that iNOS positive stained germ cells were excepted in control group 1. The statistical procedures carried out by the software of Epi INFO version 3.3 and P < 0.05 was considered statistically significant. For the comparison of the findings Kruskall–Wallis and Spearman’s correlation tests were performed. 3. Results Results were shown in Table 1. The target cell population of MPO and iNOS immunostaining is neutrophil leucocytes and mononuclear cells, respectively. The iNOS expressions in the control group were in baseline values (Fig. 1). Ischemia and reperfusion caused to increase the expression of testicular iNOS in this study (Fig. 2). In the sham operation group the mean value of iNOS immunoexpression was 26.0 ± 9.6 and the mean value of MPO immunoexpression was 38.0 ± 8.3. These values were significantly higher in T/D and T/D + saline groups. The mean immunostaining values of iNOS were 90.0 ± 17 and 95.0 ± 29.5 in T/D and T/D + saline groups, respectively. The mean immunoexpression values of MPO were179.6 ± 37.8 and 179.0 ± 38.4 in the same groups, respectively. It was demonstrated that histopathologically ischemia and reperfusion increases the expression of testicular MPO also in this study (Fig. 3). Whereas there was a significant decrease of iNOS (Fig. 4) and MPO levels in the CAPE groups. The differences of iNOS and MPO values between groups 5 and 6 were not statistically significant. But the iNOS and MPO values of in these groups were lower
E. Atik et al. / Pharmacological Research 54 (2006) 293–297
295
Table 1 Descriptive statistics of iNOS and MPO by the intervention groups (Kruskall–Wallis test) Statistical values
Intervention groups
iNOS (χ2 = 25.9, P < 0.001)
Control Sham Torsion/detorsion Torsion/detorsion + saline Torsion/detorsion + CAPE 24 h Torsion/detorsion + CAPE 48 h
n
Total
MPO (χ2 = 26.9, P < 0.001)
Control Sham Torsion/detorsion Torsion/detorsion + saline Torsion/detorsion + CAPE 24 h Torsion/detorsion + CAPE 48 h
Total
Mean
S.D.
5 5 5 5 5 5
0 26.0 90.0 95.0 66.0 62.0
0 9.6 17.0 29.5 6.5 9.0
30
56.5
37.0
5 5 5 5 5 5
0 38.0 179.6 179.0 80.0 84.0
0 8.3 37.8 38.4 11.7 17.1
30
93.4
71.3
n, the numbers of animals in each group.
Figs. 1 and 2. Fig. 1. iNOS positive stained constitutional cells in a normal testis from control group (iNOS × 400). Fig. 2. The increased amount of iNOS positive stained cells in T/D group (iNOS × 400).
Fig. 5. (A) T/D group displays a dense inflammatory infiltrate, oedema, necrosis and congestion (hematoxylin–eosine × 200). (B) The testis with normal histopathologic appearance in CAPE administrated group (hematoxylin–eosine × 400).
than groups 3 and 4. A dense inflammatory infiltrate, oedema and congestion were seen in TD group by hematoxylin–eosine staining (Fig. 5A). CAPE administration caused a histopathologically normal appearance in testis (Fig. 5B). There was a positive correlation between iNOS and MPO levels (r = 0.911 and P < 0.001 Spearman’s correlation). 4. Discussion
Figs. 3 and 4. Fig. 3. The collections of inflammatory cells positive stained by MPO in T/D group (MPO × 200). Fig. 4. Decreased number of MPO positive stained cells in CAPE administrated group (MPO × 200).
Testicular torsion is a surgical emergency. Late presentation or failure to diagnose this condition leads to testicular injury and subfertility [10,15]. Testicular injury due to torsion and detorsion is an ischemia-reperfusion (IR) injury attributed to neutrophil infiltration and generation of reactive oxygen species (ROS). In the present study we detected significantly increased iNOS and MPO values in T/D and T/D + saline groups. Current studies revealed that testicular torsion–detorsion induces histological and biochemical changes as a result of ischemia-reperfusion injury [8,10,15]. Although reperfusion is
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essential for the survival of ischemic tissue, the main pathophysiology is caused by the twisted spermatic cord and its release [8,10,15]. We demonstrated that histopathologically ischemia and reperfusion increases the expressions of testicular iNOS and MPO in this study. These high levels were compared to the control and sham groups suggest that iNOS and MPO activities may increase under abnormal conditions such as hypooxygenation. MPO is a major neutrophil protein. It is stored in azurophilic granules and released during phagocytosis [1]. MPO derived oxidants damage cells and tissue in vitro [1]. Ischemia leads to the activation of leukocytes trapped in the testicular vasculature, which may result in the release of chemotactic factors during reperfusion. Such activated neutrophils produce oxygen free radicals. These toxic oxygen derivatives cause a destructive effect on the adjacent tissue [1]. Lysiak et al. have also noted germ cell specific apoptosis after testicular IR and that an influx of neutrophils to the testis was essential for this pathology [16]. This germ cell specific apoptosis leads to a decrease in testicular weight and the loss of spermatogenesis [16]. NO is formed from l-arginine and molecular oxygen by nitric oxide synthases. Expression of iNOS requires various cytokines. The up-regulation of iNOS leads to excessive NO production and accounts for oxyradical-mediated damage in several organs. NO is an important mediator of cell death either through apoptosis or necrosis [5]. Otherwise sustained production of NO by iNOS in response to inflammatory stimuli has been shown to mediate antimicrobial and antitumoral activities [7]. Paradoxically oxygen is both essential for life as well as, toxic to human. It has been demonstrated that free oxygen radicals causing injury in post-ischemic cells and tissue with a critical mechanism [11]. Testicular injury due to T/D is an ischemia-reperfusion injury attributed to neutrophil infiltration, generation of ROS and reactive nitrogen species. ROS and reactive nitrogen species such as nitric oxide cause DNA damage. DNA damage can lead to loss of cell viability [3,10,11,15–17]. Ischemia causes an increase in NOS activity in the vascular endothelium and, later in a range of cells including infiltrating neutrophils and macrophages. While NO levels increase during ischemia, reperfusion leads to the generation of the superoxide radicals through the action of several mechanisms; the interaction between NO and O2 produces peroxynitrite. The breakdown of NO by O2 anions may be a factor in the decreased NO levels [8,17]. We found that increased expression of iNOS in T/D and T/D + saline groups, which is consistent with literature [17]. However, NO levels were a little increased in ischemia but decreased in reperfusion according to studies of Uz et al. [1] and Koltuksuz et al. [8]. Es¸refo˘glu et al. have demonstrated that the role of oxidative injury on testicular damage following myocardial IR injury and the increase of NO. They also noted inhibition of NO by CAPE and prevented testicular damage [17]. Despite intensive research, the role of NO remains uncertain in post-ischemic tissue injury and NO production is increased or decreased?
In another study XO was reported to catalyze the reduction of NO3 and NO2 to NO under hypoxic conditions [8]. We also demonstrated that CAPE administration brought the iNOS and MPO immunoexpressions around the sham values. CAPE administration attenuates the increase in the tissue MPO and iNOS levels. As a result we can say, – the beneficial effect of CAPE on testicular damage after T/D may be depend on CAPE’s antineutrophil effects – prophylactic CAPE administration in testicular ischemia may prevent reperfusion injury by eliminating reactive nitrogen species. We demonstrated that T/D increases the immunoexpression values of testicular iNOS and MPO. The administration of CAPE prevents the increase in immunoexpression values of iNOS and MPO, thus CAPE may be useful in dealing with ischemiareperfusion injuries. These results partially explain the pathophysiology of testis injury after IR and suggest that inhibition of iNOS and MPO by CAPE may be a novel pharmacological approach to prevent cell injury. However, further studies are required to clarify the exact mechanism of IR injury and effects of CAPE. Acknowledgement We are very grateful to Dr. Tacettin ˙Inandı for his kindly helping about statistical analyse. References ¨ [1] Uz E, S¨og˘ u¨ t S, S¸ahin S¸, Var A, Ozyurt H, G¨ulec¸ M, et al. The protective role of caffeic acid phenethyl ester (CAPE) on testicular tissue after testicular torsion and detorsion. World J Urol 2002;20:264–70. [2] Romeo C, Antonuccio P, Esposito M, Marini H, Impellizzeri P, Turiaco N, et al. Raxofelast, a hydrophilic vitamin e-like antioxidant, reduces testicular ischemia-reperfusion injury. Urol Res 2004;32:367–71. [3] Uguralp S, Karabulut AB, Mızrak B, Kaymaz F, Kızıltay F, Hasirci N. The effect of sustained and local administration of epidermal growth factor on improving bilateral testicular tissue after torsion. Urol Res 2004;32:323–31. [4] Lysiak JJ, Turner SD, Nguyen QA, Singbartl K, Ley K, Turner TT. Essential role of neutrophils in germ cell-specific apoptosis following ischemia/reperfusion injury of the mouse testis. Biol Reprod 2001;65:718–25. [5] Shiraishi K, Naito K, Yoshida Ki. Nitric oxide promotes germ cell necrosis in the delayed phase after experimental testicular torsion of rat. Biol Reprod 2001;65:514–21. [6] Fujisawa M, Tatsumi N, Fujioka H, Kanzaki M, Okuda Y, Arakawa S. Nitric oxide production of rat Leydig and Sertoli cells is stimulated by round spermatic factors. Mol Cell Endocrinol 2000;160:99–105. [7] Bauche F, Stephan JP, Touzalin AM, Jegou B. In vitro regulation of an inducible-type NO synthase in the rat seminiferous tubule cells. Biol Reprod 1998;58:431–8. ¨ [8] Koltuksuz U, Irmak MK, Karaman A, Uz E, Var A, Ozyurt H, et al. Testicular nitric oxide levels after unilateral testicular torsion/detorsion in rats pretreated with caffeic acid phenethyl ester. Urol Res 2000;28:360–3. [9] O’Bryan MK, Schlatt S, Gerdprasert O, Phillips DJ, Kretser DM, Hedger MP. Inducible nitric oxide synthase in the rat testis: evidence for potential roles in both normal function and inflammation-mediated infertility. Biol Reprod 2000;63:1285–93. [10] Bozlu M, Eskandari G, C¸ayan S, Canpolat B, Akbay E, Atik U, et al. The effect of poly(adenosine diphosphate-ribose) polymerase inhibitors
E. Atik et al. / Pharmacological Research 54 (2006) 293–297 on biochemical changes in testicular ischemia-reperfusion injury. J Urol 2003;169:1870–3. ¨ T¨urk¨oz Y, Hasc¸alık S¸, Hasc¸alık M, Cigremis Y, Mızrak B, et [11] C¸elik O, al. The protective effect of caffeic acid phenethyl ester on ischemiareperfusion injury in rat ovary. Eur J Obstet Gyn R B 2004;117(2): 183–8. ¨ ¨ Iraz M, S¨og˘ u¨ t S, Ozu˘ ¨ gurlu F, Ozyurt ¨ [12] Ozen S, Akyol O, F, et al. Role of caffeic acid phenethyl ester, an active component of propolis, against cisplatin-induced nephrotoxicity in rats. J Appl Toxicol 2004;24: 27–35. [13] Sud’ina GF, Mirzoeva OK, Pushkareva MA, Korshunova GA, Sumbatyan NV, Varfolomeev SD. Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett 1993;329:21–4.
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[14] Grunberger D, Banerjee R, Eisinger K, Oltz EM, Efros L, Caldwell M, et al. Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis. Experientia 1988;44:230–2. [15] Bozlu M, Cos¸kun B, C¸ayan S, Acar D, Aktas¸ S, Ulusoy E, et al. Inhibition of poly(adenosine diphosphate-ribose) polymerase decreases long-term histologic damage in testicular ischemia-reperfusion injury. Urology 2004;63:791–5. [16] Lysiak JJ, Nguyen QAT, Turner TT. Peptide and non-peptide reactive oxygen scavengers provide partial rescue of the testis after torsion. J Androl 2002;23:400–9. [17] Es¸refo˘glu M, G¨ul M, Parlakpınar H, Acet A. Effects of melatonin and caffeic acid phenethyl ester on testicular injury induced by myocardial ischemia/reperfusion in rats. Fundam Clin Pharmacol 2005;19:365–72.