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Coenzyme Q10 treatment reduces lipid peroxidation, inducible and endothelial nitric oxide synthases, and germ cell–specific apoptosis in a rat model of testicular ischemia/reperfusion injury

2009; Elsevier BV; Volume: 93; Issue: 1 Linguagem: Inglês

10.1016/j.fertnstert.2009.07.981

1556-5653

Bülent Erol, Murat Bozlu, Volkan Hancı, Hüsnü Tokgöz, Sibel Bektaş, Görkem Mungan,

Aldose Reductase and Taurine

In this experimental study, we assessed the preventive effects of coenzyme Q10 (CoQ10) in a rat model of ischemia/reperfusion injury. The results of this study show that CoQ10 administration before the reperfusion period of testicular torsion provides a significant decrease in testicular lipid peroxidation products and expressions of inducible nitric oxide synthase, endothelial nitric oxide synthase, and germ cell-specific apoptosis. In this experimental study, we assessed the preventive effects of coenzyme Q10 (CoQ10) in a rat model of ischemia/reperfusion injury. The results of this study show that CoQ10 administration before the reperfusion period of testicular torsion provides a significant decrease in testicular lipid peroxidation products and expressions of inducible nitric oxide synthase, endothelial nitric oxide synthase, and germ cell-specific apoptosis. Testicular torsion is a urologic emergency that causes testicular injury and subfertility (1Visser A.J. Heyns C.F. Testicular function after torsion of the spermatic cord.BJU Int. 2003; 92: 200-203Crossref PubMed Scopus (196) Google Scholar). It appears that the main pathophysiology of testicular torsion is ischemia/reperfusion (I/R) injury of the testis caused by the twisted spermatic cord and its release (2Cuzzocrea S. Riley D.P. Caputi A.P. Salvemini D. Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/reperfusion injury.Pharmacol Rev. 2001; 53: 135-159PubMed Google Scholar, 3Lysiak J.J. Nguyen Q.A. Kirby J.L. Turner T.T. Ischemia-reperfusion of the murine testis stimulates the expression of proinflammatory cytokines and activation of c-jun N-terminal kinase in a pathway to E-selectin expression.Biol Reprod. 2003; 69: 202-210Crossref PubMed Scopus (121) Google Scholar). Although reperfusion is essential for the survival of ischemic tissue, there is good evidence that reperfusion itself causes additional cell injury. Coenzyme Q10 (CoQ10) is an essential component for electron transport in oxidative phosphorylation of mitochondria (4Littarru G.P. Tiano L. Clinical aspects of coenzyme Q10: an update.Curr Opin Clin Nutr Metab Care. 2005; 8: 641-646Crossref PubMed Scopus (88) Google Scholar, 5Belardinelli R. Tiano L. Littarru G.P. Oxidative stress, endothelial function and coenzyme Q10.Biofactors. 2008; 32: 129-133Crossref PubMed Scopus (36) Google Scholar). It is a potent antioxidant, a membrane stabilizer, and cofactor in the production of adenosine triphosphate by oxidative phosphorylation. Coenzyme Q10 has been widely applied in food supplements and cosmetics in Japan, the U.S., and many other countries. In recent years, the frequency of studies involving CoQ10 has increased in both basic and clinical research areas (4Littarru G.P. Tiano L. Clinical aspects of coenzyme Q10: an update.Curr Opin Clin Nutr Metab Care. 2005; 8: 641-646Crossref PubMed Scopus (88) Google Scholar, 5Belardinelli R. Tiano L. Littarru G.P. Oxidative stress, endothelial function and coenzyme Q10.Biofactors. 2008; 32: 129-133Crossref PubMed Scopus (36) Google Scholar, 6Hatanaka J. Kimura Y. Lai-Fu Z. Onoue S. Yamada S. Physicochemical and pharmackinetic characterization of water-soluble coenzyme Q10 formulations.Int J Pharm. 2008; 363: 112-117Crossref PubMed Scopus (82) Google Scholar, 7Miles M.V. The uptake and distribution of coenzyme Q(10).Mitochondrion. 2007; 7S: 72-77Crossref Scopus (103) Google Scholar). Several studies have demonstrated the protective effect of CoQ10 in various forms of tissue injury (4Littarru G.P. Tiano L. Clinical aspects of coenzyme Q10: an update.Curr Opin Clin Nutr Metab Care. 2005; 8: 641-646Crossref PubMed Scopus (88) Google Scholar). However, no study has investigated the effect of CoQ10 in testicular I/R injury. The present study aimed to assess the effects of CoQ10 administration on lipid peroxidation, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and germ cell–specific apoptosis in a rat model of testicular I/R injury. The study included 21 adult male Wistar rats weighing 240–260 g. The rats were maintained on a 12-hour light/dark cycle after ethical committee on animal research approval. Institutional Review Board approval was obtained. The rats were divided into three groups of seven rats each. One group underwent 1 hour of testicular torsion, the second received pretreatment with CoQ10 before detorsion, and the third underwent a sham operation. Surgery was done with the subject under ketamine anesthesia (single intraperitoneal 50 mg/kg dose). All surgical procedures were performed through standard ilioinguinal incisions (8Bozlu M. Eskandari G. Cayan S. Canpolat B. Akbay E. Atik U. The effect of poly(adenosine diphosphate-ribose) polymerase inhibitors on biochemical changes in testicular ischemia-reperfusion injury.J Urol. 2003; 169: 1870-1873Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). At the end of the experiments, bilateral orchiectomies were performed and the rats were killed by pentobarbital overdose (200 mg/kg) and bilateral thoracotomy. In the torsion-detorsion (T-D) group, the gubernaculum was divided, and the testis was freed from its longitudinal and distal pole attachement to the epididymis. Torsion was created by rotating the left testis 720° clockwise and maintained by fixing the testis to the scrotum with a 4-0 silk suture placed through the tunica albuginea. After 1 hour of torsion, the testis was counter-rotated back to the natural position and reinserted into the scrotum. The wound was closed using 3-0 catgut suture. After detorsion for 4 hours, bilateral orchiectomies were performed. In the group receiving CoQ10 before detorsion, the same surgical procedure was done as in the T-D group, but CoQ10 (10 mg/kg; Sigma Chemical Co., St. Louis, MO) was injected intraperitoneally for 30 minutes before detorsion. The administration mode and dose of CoQ10 corresponded to those used in earlier experimental studies (7Miles M.V. The uptake and distribution of coenzyme Q(10).Mitochondrion. 2007; 7S: 72-77Crossref Scopus (103) Google Scholar). After detorsion for 4 hours, bilateral orchiectomies were performed. In the control group (sham group), a sham procedure was performed. This consisted of same procedure as in the T-D group, except that after rotating the testis 720° clockwise, it was immediately relieved, and a 4-0 silk suture was placed through the tunica albuginea. Bilateral orchiectomies were performed after the procedure. Testicular lipid peroxidation was estimated by the measurement of malondialdehyde (MDA) (9Esterbauer H. Schaur R.J. Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes.Free Radic Biol Med. 1991; 11: 81-128Crossref PubMed Scopus (5821) Google Scholar). High-performance liquid chromatographic (HPLC) analysis was performed using a Shimadzu HPLC system (Kyoto, Japan) with MDA kit (Immundiagnostik, Bensheim, Germany). Immunohistochemical evaluation of iNOS, eNOS, and germ cell–specific apoptosis were performed with iNOS Ab-1, eNOS Ab-1, and apoptosis protease–activating factor 1 (APAF-1) antibody (Lab Vision Corp., Neomarkers, CA), respectively (10Yagmurdur H. Ayyildiz A. Karaguzel E. Akgul T. Ustun H. Germiyanoglu C. Propofol reduces nitric oxide–induced apoptosis in testicular ischemia-reperfusion injury by downregulating the expression of inducible nitric oxide synthase.Acta Anaesthesiol Scand. 2008; 52: 350-357Crossref PubMed Scopus (29) Google Scholar). The number of stained germ cells were counted in 100 seminiferous tubules on circular cross-sections for each group. All data are expressed as mean ± SD. Analysis of variance was used for statistical analysis of the data among the groups. Multiple comparisons were made using Tukey procedure, with P<.05 considered to be statistically significant. The values of testicular MDA levels and iNOS, eNOS, and APAF-1 expressions of each group are shown in Table 1. Compared with the sham group, testicular MDA levels and expressions of iNOS, eNOS, and APAF-1 obtained from the T-D group were significanlty higher in the ipsilateral testes (P<.001). Ipsilateral testicular MDA levels and iNOS, eNOS, and APAF-1 expressions obtained from the CoQ10 treatment group were significantly lower than those obtained from the T-D group (P<.01). All of the parameters of contralateral testes did not reveal any statistically significant differences among the groups (P>.05).Table 1The values of MDA levels and iNOS, eNOS and APAF-1 expressions in ipsilateral and contralateral testes (mean ± SD).MDA (μmol/g protein)iNOS (positive staining/tubule)eNOS (positive staining/tubule)APAF-1 (positive staining/tubule)GroupIpsilateralContralateralIpsilateralContralateralIpsilateralContralateralIpsilateralContralateralTorsion-detorsionaVersus sham in the ipsilateral testes: P<.001.1.99 ± 0.450.38 ± 0.125.88 ± 2.011.15 ± 0.856.55 ± 3.081.12 ± 0.5425.62 ± 6.3910.15 ± 1.22CoQ10bVersus torsion-detorsion in the ipsilateral testes: P<.01.0.71 ± 0.210.4 ± 0.182.57 ± 0.781.13 ± 0.753.28 ± 0.951.16 ± 0.6211.00 ± 1.159.95 ± 1.18Sham0.42 ± 0.150.39 ± 0.11.12 ± 0.921.15 ± 0.951.18 ± 0.421.14 ± 0.79.28 ± 0.959.75 ± 1.15Note: There were no statistically significant differences in the contralateral testes among the groups (P>.05).APAF-1 = apoptosis protease–activating factor 1; CoQ10 =; coenzyme Q10; eNOS = endothelial nitric oxide synthase; iNOS = inducible nitric oxide synthase; MDA = malondialdehyde.a Versus sham in the ipsilateral testes: P<.001.b Versus torsion-detorsion in the ipsilateral testes: P<.01. Open table in a new tab Note: There were no statistically significant differences in the contralateral testes among the groups (P>.05). APAF-1 = apoptosis protease–activating factor 1; CoQ10 =; coenzyme Q10; eNOS = endothelial nitric oxide synthase; iNOS = inducible nitric oxide synthase; MDA = malondialdehyde. In agreement with earlier studies (8Bozlu M. Eskandari G. Cayan S. Canpolat B. Akbay E. Atik U. The effect of poly(adenosine diphosphate-ribose) polymerase inhibitors on biochemical changes in testicular ischemia-reperfusion injury.J Urol. 2003; 169: 1870-1873Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 11Turner T.T. Tung K.S. Tomomasa H. Wilson L.W. Acute testicular ischemia results in germ cell–specific apoptosis in the rat.Biol Reprod. 1997; 57: 1267-1274Crossref PubMed Scopus (258) Google Scholar, 12Shirashi K. Naito K. Yoshida K. Nitric oxide promotes germ cell necrosis in the delayed phase after experimental teseticular torsion of rat.Biol Reprod. 2001; 65: 514-521Crossref PubMed Scopus (78) Google Scholar), we found that testicular T-D caused a significant increase in testicular lipid peroxidation products and expressions of iNOS and eNOS and germ cell–specific apoptosis. The results from the present study indicated that CoQ10 administration before the reperfusion period of testicular torsion provided a significant decrease in testicular lipid peroxidation products and expressions of iNOS, eNOS, and germ cell–specific apoptosis. Coenzyme Q10 was first introduced as an ethical drug for heart failure patients in Japan and other nations. Coenzyme Q10, which functions endogenously in the mitochondrial electron transport chain, can be ingested to scavenge free radicals and contribute to antioxidant defenses in vivo (5Belardinelli R. Tiano L. Littarru G.P. Oxidative stress, endothelial function and coenzyme Q10.Biofactors. 2008; 32: 129-133Crossref PubMed Scopus (36) Google Scholar). In recent years, the role of CoQ10 in disease prevention and treatment has been intensely investigated (4Littarru G.P. Tiano L. Clinical aspects of coenzyme Q10: an update.Curr Opin Clin Nutr Metab Care. 2005; 8: 641-646Crossref PubMed Scopus (88) Google Scholar). The successful results of CoQ10 administration in different organ systems led us to attempt such treatment with a model of testicular I/R injury. To our knowledge, the effect of CoQ10 on testicular I/R injury has not been reported. We demonstrated that administration of CoQ10 treatment before reperfusion caused significant reduction in the level of testicular MDA. It has been demonstrated that CoQ10 functions as an antioxidant, scavenging free radicals and inhibiting lipid peroxidation (5Belardinelli R. Tiano L. Littarru G.P. Oxidative stress, endothelial function and coenzyme Q10.Biofactors. 2008; 32: 129-133Crossref PubMed Scopus (36) Google Scholar). The present data indicate that treatment with CoQ10 prevents further increase in lipid peroxidation caused by I/R; therefore, it may protect the testis against oxidative damage. The results of earlier studies suggest that NO has an important role in damage of the testis via I/R (8Bozlu M. Eskandari G. Cayan S. Canpolat B. Akbay E. Atik U. The effect of poly(adenosine diphosphate-ribose) polymerase inhibitors on biochemical changes in testicular ischemia-reperfusion injury.J Urol. 2003; 169: 1870-1873Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 12Shirashi K. Naito K. Yoshida K. Nitric oxide promotes germ cell necrosis in the delayed phase after experimental teseticular torsion of rat.Biol Reprod. 2001; 65: 514-521Crossref PubMed Scopus (78) Google Scholar). It has been investigated in several organs that after ischemia, superoxide is produced during the reperfusion phase which rapidly reacts with NO and forms reactive nitrogen species peroxynitrite (2Cuzzocrea S. Riley D.P. Caputi A.P. Salvemini D. Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/reperfusion injury.Pharmacol Rev. 2001; 53: 135-159PubMed Google Scholar). Peroxynitrite initiates toxic oxidative reactions, directly inhibits mitocondrial respiratory enzymes, decreases cellular oxygen consumption, and inhibits membrane sodium transport. Coenzyme Q10 can reverse endothelial dysfunction by preventing oxidative and nitrative stress and inflammation (5Belardinelli R. Tiano L. Littarru G.P. Oxidative stress, endothelial function and coenzyme Q10.Biofactors. 2008; 32: 129-133Crossref PubMed Scopus (36) Google Scholar). It may act to scavenge oxidant species, thereby reducing oxidative stress and resulting in recoupling of NOS (13Chew G.T. Watts G.F. Coenzyme Q10 and diabetic endotheliopathy: oxidative stress and the "recoupling hypothesis".QJM. 2004; 97: 537-548Crossref PubMed Scopus (103) Google Scholar). We demonstrated that treatment with CoQ10 significantly decreases the expressions of iNOS and eNOS. In the light of these data, one may anticipate that administrating CoQ10 before reperfusion would protect the testis against NO-related injury in I/R. Turner et al. (11Turner T.T. Tung K.S. Tomomasa H. Wilson L.W. Acute testicular ischemia results in germ cell–specific apoptosis in the rat.Biol Reprod. 1997; 57: 1267-1274Crossref PubMed Scopus (258) Google Scholar) reported that testicular T-D in the rat resulted in germ cell–specific apoptosis. In the present study, we showed that T-D caused a significant increase in APAF-1 expression in the testis. APAF-1 plays a central role in mitochondrial apoptosis (14Zuo Y. Xiang B. Yang J. Sun X. Wang Y. Chang H. et al.Oxidative modification of caspase-9 facilitates its activation via disulfide-mediated interaction with APAF-1.Cell Res. 2009; 19: 449-457Crossref PubMed Scopus (82) Google Scholar). The release of cytochrome c from the mitochondria leads to recruitment of procaspase 9 and APAF-1 into a protein complex that subsequently activates caspase 9. Lysiak et al. (15Lysiak J.J. Zheng S. Woodson R. Turner T.T. Caspase-9–dependent pathway to murine germ cell apoptosis: mediation by oxidative stress, BAX, and caspase 2.Cell Tissue Res. 2007; 328: 411-419Crossref PubMed Scopus (56) Google Scholar) found that IR of the testis stimulated a mitochondrial- and caspase 9–dependent pathway to germ cell–specific apoptosis. To our knowledge, the role of CoQ10 on germ cell–specific apoptosis of testis induced by I/R is undefined. Under our experimental conditions, we found that administering CoQ10 before reperfusion caused a significant decrease in the testicular APAF-1 expression compared with that in the T-D group. Recent investigations have emphasized the antiapoptotic effect of CoQ10 and the protective effect of CoQ10 against apoptotic changes in several tissues and cells (16Witort E. Pattarino J. Papucci L. Schiavone N. Donnini M. Lapucci A. et al.Autologous lipofilling: coenzyme Q10 can rescue adipocytes from stress-induced apoptotic death.Plast Reconstr Surg. 2007; 119: 1191-1199Crossref PubMed Scopus (27) Google Scholar, 17Wu K.L.H. Hsu C. Chan J.Y.H. Impairment of the mitochondrial respiratory enzyme activity triggers sequential activation of apoptosis-inducing factor–dependent and caspase-dependent signaling pathways to induce apoptosis after spinal cord injury.J Neurochem. 2007; 101: 1552-1566Crossref PubMed Scopus (40) Google Scholar). Emerging evidence indicates that a central event of apoptosis is opening of the mitocondrial permeability transition pore. Opening of the permeability transition pore is responsible for disruption of the mitochondrial transmembrane electrochemical gradient and is accompanied by extrusion to cytoplasm of several molecules, including cytochrome c and APAF-1, responsible for caspase cascade activation. The antiapoptotic activity of CoQ10 is mediated by hindering mitochondrial depolarization, cytochrome c release to cytoplasm, and activation of caspase 9 (16Witort E. Pattarino J. Papucci L. Schiavone N. Donnini M. Lapucci A. et al.Autologous lipofilling: coenzyme Q10 can rescue adipocytes from stress-induced apoptotic death.Plast Reconstr Surg. 2007; 119: 1191-1199Crossref PubMed Scopus (27) Google Scholar, 17Wu K.L.H. Hsu C. Chan J.Y.H. Impairment of the mitochondrial respiratory enzyme activity triggers sequential activation of apoptosis-inducing factor–dependent and caspase-dependent signaling pathways to induce apoptosis after spinal cord injury.J Neurochem. 2007; 101: 1552-1566Crossref PubMed Scopus (40) Google Scholar). Coenzyme Q10 may be endowed with antiapoptotic activity as a modulator of permeability transition pore opening. On the other hand, we demonstrated that CoQ10 may confer protective effect against lipid peroxidation in testicular I/R injury, and germ cell–specific apoptosis may be suppressed by this antioxidant action of CoQ10. However, this study is the first report in the literature to show that the apoptotic changes seen in germ cells were reduced by CoQ10 in the rat model of testicular torsion. To date, several enzymes and drugs have been studied to alleviate testicular damage in animal models of I/R injury (1Visser A.J. Heyns C.F. Testicular function after torsion of the spermatic cord.BJU Int. 2003; 92: 200-203Crossref PubMed Scopus (196) Google Scholar, 10Yagmurdur H. Ayyildiz A. Karaguzel E. Akgul T. Ustun H. Germiyanoglu C. Propofol reduces nitric oxide–induced apoptosis in testicular ischemia-reperfusion injury by downregulating the expression of inducible nitric oxide synthase.Acta Anaesthesiol Scand. 2008; 52: 350-357Crossref PubMed Scopus (29) Google Scholar, 18Dokmeci D. Kanter M. Inan M. Aydogdu N. Basaran U.N. Yalcin O. Turan F.N. Protective effects of ibuprofen on testicular torsion/detorsion–induced ischemia/reperfusion injury in rats.Arch Toxicol. 2007; 81: 655-663Crossref PubMed Scopus (48) Google Scholar). However, none have been tested in clinical trials, apart from cooling the scrotum. Coenzyme Q10 was first introduced as a drug for heart failure patients; its present status in most countries is that of a compound aimed at improving cellular bioenergetics, counteracting oxidative stress, and slowing down some age-related pathologies; it is also used as a preventive support measure. In conclusion, the use of CoQ10 in humans in both earlier studies and clinically without significant side effects can make its potential use in testicular torsion more attractive. The results of the present experimental study show that administration of CoQ10 may be a novel approach for the therapy of I/R injury of the testis.