Exp. Anim. 60(5), 445–453, 2011

—Original— Quinestrol Treatment Induced Testicular Damage via Oxidative Stress in Male Mongolian Gerbils (Meriones unguiculatus)

Wei SHEN1), Dazhao SHI1), Deng WAND1), Yongwang GUO2), Shuzhen HAI1), and Zhuo YUE3)

1)College of Agriculture and Biotechnology, Agricultural University, Beijing 100193, 2)National Agro-tech Extensions and Service Center, Beijing 100125, and 3)College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

Abstract: The hypothesis that quinestrol exerts testicular damage via oxidative stress was investigated in male gerbils using a daily oral gavage of 3.5 mg/kg body weight for 2 weeks (the multidose-treated group) or 35 mg/kg body weight (the single-dose-treated group). The testicular histological morphology, antioxidant capacity and malondialdehyde (MDA) concentration in testicular tissue and plasma were assessed at 15, 30, and 60 days following treatment. The results showed that the activity of the antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxide (GSH-Px), and total antioxidant capacity (T-AOC), at 15 days after treatment in testicular tissue decreased, which led to the MDA concentration increasing while at the same time germ cells were rarefied and showed an irregular distribution in seminiferous tubules of quinestrol-treated gerbils. At 30 days, the testicular weight and antioxidant capacity continued to decrease, while the MDA concentration continued to increase, and testicular histopathological changes were more pronounced. Single-dose and multidose drug treatment had a similar effect on the antioxidant enzymes and MDA, but testicular damage was relatively severe at 15 and 30 days after multidose treatment. By 60 days of treatment withdrawal, however, the above parameters recovered to control levels. The results show that quinestrol causes reversible damage to gerbil testes that might be caused by the oxidative stress and that multidose treatment has more effects on testicular damage compared with one-dose treatment. Key words: Mongolian gerbil (Meriones unguiculatus), oxidative damage, quinestrol, reversibility, testicular damage

Introduction Subsequently, both laboratory and field experiments showed that EP-1 and its individual components, Levonorgestrel-Quinestrol (Code: EP-1) was first used levonorgestrel and quinestrol, affected the structure of as a new type of rodenticide by Zhibin Zhang of the reproductive organs and exerted antifertility effects in Institute of Zoology, Chinese Academy of Sciences [31]. many wild rodent species, such as the greater long-tailed

(Received 18 January 2011 / Accepted 10 May 2011) Address corresponding: D. Shi, College of Agriculture and Biotechnology, China Agricultural University, No.2 Yuanmingyuan West Road, Haidian District, Beijing 100193, China 446 W. Shen, ET AL. hamster (Tscherskia triton) [32, 33], Mongolian gerbils weight as well as histological changes in testes and (2) (Meriones unguiculatus) [13, 14] and the Djungarian the effect of quinestrol on the levels of MDA, SOD, hamster (Phodopus campbelli) [30]. According to GSH-Px, and T-AOC in vivo. Meirong Zhao (2007), quinestrol is an effective inducer of EP-1 in male reproductive damage [34]. Materials and Methods Quinestrol (17-alpha -3-cyclopentyl ether), a derivative of ethinylestradiol, is a long-acting Animals and quinestrol treatment synthetic . Its estrogenic activity is about 8 to Forty-five adult male gerbils (4–6 months old, average 10 times that of its matrix, and it has strong antifertility weight 56.05 ± 7.94 g) captured from farmland in Kang- effects (http://en.wikipedia.org/wiki/Quinestrol). Ani- bao County, Hebei Province, were used in this study. mal experiments show that after oral administration, The animals were housed in individual polypropylene quinestrol is absorbed by the gastrointestinal tract and cages with a 12-h light: 12-h dark cycle at 20 to 21ºC. stored in the ; then it is gradually released All animals had ad libitum access to corn, sunflower into the bloodstream at low levels to maintain a long- seeds, and water in glass bottles with rubber stoppers lasting effective concentration [19, 20]. and were provided with cabbage every three days. After Quinestrol affects secretion via inhibi- 1 week of acclimation, they were randomly divided into tion of the hypothalamus-pituitary-ovarian axis. Quin- 3 groups (15 animals per group), including the control estrol-treated females are infertile with abnormal folli- group, the single-dose-treated group (the SDT group), culogenesis. Recently, much attention has been focused and multi-dose-treated group (the MDT group). There on oxidative stress as a causal factor of male infertility was no significant difference in the body weights of the

[3]. Reactive oxygen species (ROS) are produced by animals in each group (F(2, 42) = 0.087, P>0.05). The use aerobic cellular metabolism and can damage normal of animals in this study was approved by the Chinese physiological functions of biological macromolecules Association for Laboratory Animal Sciences. such as the plasma membrane, carbohydrates and pro- According to the results of our a preparative study, teins. Different concentrations of ROS can induce apop- administration of single peanut oil or multiple of peanut tosis or cell necrosis [21]. The testicular tissue and oil has no effect on the body weight and reproductive sperm plasma membrane, which is rich in polyunsatu- organs of gerbils. So the control group was given 35 mg rated fatty acids, are highly susceptible to ROS damage of peanut oil/kg body weight only once. The MDT group [6]. ROS and lipid peroxidation (LPO) are known to be was given 3.5 mg of quinestrol/kg body weight for 5 the vital mediators in testis physiology [17, 29]. SOD consecutive days and then for another 5 days after a and GSH-PX are the most important antioxidant enzymes 2-day interval. The SDT group was given 35 mg of to protect cells from oxidative stress [9]. Quinestrol is quinestrol/kg body weight only once. Five animals per known to cause infertility in males by inducing testicu- group were collected for analysis at 15, 30, or 60 days lar damage [13, 30–34]. However, as far as we know, after the first of treatment. The males were weighed, few studies have examined whether quinestrol induces and blood samples were collected from the infraorbital infertility involving oxidative stress damage and the venous plexus. After centrifugation, the serum was reversibility of such damage. Thus, the aims of the pres- stored at –20°C until the antioxidant enzyme analysis. ent study were to test the hypothesis that quinestrol After sacrificing the animals by cervical dislocation, the exerts its toxicity in the testis by causing oxidative dam- right and left testes were immediately collected and age and to test its reversibility. weighed. Relative testicular weights were expressed as Here, we evaluated the testicular toxicity of quinestrol the weight of the testes (g)/body weight (kg). The right in male gerbils by administration of two different doses. testis of each gerbil was immediately homogenized in The dose levels of this experiment were based on the ice-cold PBS (pH 7.4).The homogenate was centrifuged results of our preparative study in gerbils. Specifically, at 3,000 × g for 15 min, and the supernatant was col- we examined (1) the effect of quinestrol on testicular lected to assay for antioxidant enzymes. Quinestrol Induced Testicular Damage 447

Histology analysis Results The left testes were fixed in 2.5% glutaraldehyde- paraformaldehyde solution for 24 h. After fixing, the Gross changes in testes tissues were dehydrated in ethanol and embedded in At 15 and 30 days after quinestrol treatment, the tes- paraffin. Five-micron sections were collected on glass tes were significantly atrophic. The relative testicular slides, and routine hematoxylin and eosin (HE) staining weights of each gerbil of the SDT and MDT groups at was performed. 15 days were 6.704 (P<0.001) and 6.180 (P<0.001), respectively, compared with that of the control gerbils Assessment of antioxidant activity (17.011, Fig. 1, Table 1). At 30 days, the relative tes- GSH-Px activity was measured according to the ticular weights were significantly different between the method of Annamarie Drotar [6] using glutathione as the MDT (4.368) and SDT groups (8.815, P<0.05). At 60 substrate. GSH-Px could promote the reaction of hy- days, the testicular morphology and size were restored drogen peroxide (H2O2) and glutathione (GSH) to H2O such that they were similar to those of the control group and oxidized glutathione (GSSG). The decrease rate of (Fig. 1, Table 1). Therefore, quinestrol had a significant GSH could be used to express GSH-Px activity. GSH effect on testes, and its damage to the testes in the MDT content was assayed according to the formation rate of group was more severe than that of the SDT group at 30 5-thio-2-nitrobenzoic acid (TNB) reduced from DTNB days. However, quinestrol-induced damage was reversed with 412-nm absorbance. The assays for total SOD were upon drug withdrawal. based on the ability to inhibit the oxidation of oxyamine by the xanthine–xanthine oxidase system [22]. The red Histological changes in seminiferous tubules product (nitrite) produced by the oxidation of oxyamine In the control group, the structure of the testis was had absorbance at 550 nm. The lipid peroxidation prod- ucts (measured as MDA) were measured by the thiobar- bituric acid (TBA) colorimetric method [25], and optical Table 1. Comparison of testicular weights, relative testicular density was measured at 532 nm. T-AOC levels were weights, and body weight at 15, 30, and 60 days after treatment with quinestrol based on the ability to deoxidize Fe3+ to Fe2+, and optical Testicular weight (g)* density was measured at 520 nm. The protein concentra- Days 15 30 60 tion was determined using a standard commercial kit. SDT 0.418 ± 0.213a) 0.520 ± 0.261d) 0.863 ± 0.229b) Values were expressed as units (U) per mg protein for MDT 0.330 ± 0.268a) 0.244 ± 0.061c) 0.817 ± 0.168b) SOD, GSH-Px, T-AOC, and nmol/mg protein for MDA. CK 0.940 ± 0.321b) 0.924 ± 0.201b) 0.961 ± 0.232b) Determinations and procedures were performed accord- ing to the recommended procedures provided by the Relative testicular weight (g/kg body weight)* commercial kits (Jiancheng Institute of Biotechnology, Days 15 30 60 Nanjing, China). SDT 6.704 ± 3.154a) 8.815 ± 2.586d) 13.532 ± 2.431b) MDT 6.180 ± 3.170a) 4.368 ± 1.356c) 14.194 ± 1.975b) Statistics CK 17.011 ± 3.045b) 16.513 ± 3.045b) 15.504 ± 2.241b) The differences between various groups were analyzed using one-way analysis of variance (ANOVA) by a mul- Final body weight (g)* tivariate comparison procedure using SPSS 12.0 soft- Days 15 30 60 ware. The differences between two groups were ana- SDT 57.12 ± 4.15 57.83 ± 4.54 58.21 ± 5.24 lyzed using the Student’s t-test. Results were considered MDT 57.25 ± 5.34 58.54 ± 5.27 56.95 ± 6.46 CK 58.54 ± 5.04 57.14 ± 4.15 57.54 ± 5.43 to be statistically significant at P<0.05. The data are expressed as means±SD. *Results are expressed as means ± SD. The mean differences between the values bearing different superscript letters within the same column or row are statistically significant (a), b), c), and d): P<0.05). 448 W. SHEN, ET AL.

Fig. 1. Morphological changes in the testes at 30 days (A) and 60 days (B) after quinestrol treatment.

Fig. 2. Histological changes in the testes at 15, 30, and 60 days after treatment with quinestrol (HE stain, × 200 magnifi cation) (A and B) The SDT group at 15 and 30 days and (E) MDT group at 15 days after treatment with quinestrol showed that some seminiferous tubules were normal and contained sperm. (D) In the control group, the structure of the seminiferous tubules was clear and fi lled with numerous sperms. (F) In the MDT group at 30 days after treatment, germ cells in the seminiferous tubules were scattered irregularly, and almost no sperm were observed in the lumen. (C and G) The abnormal seminiferous tubules were normal at 60 days after the fi rst treatment.

intact. The seminiferous tubules were closely arranged. At 30 days, the damage was increased (Fig. 2B). Many The testis structure was clear, and spermatogenetic cells spermatogenetic cells had undergone necrosis, and the were tightly and orderly arranged in lumen. Sperm cells seminiferous epithelium was detached from the basement were dense, and a large number of sperm were in the membrane. The proportions of spermatogonia, sperma- lumen (Fig. 2D). At 15 days, only slight pathological tocytes, and sperm cells were obviously different from changes were observed in the SDT group (Fig. 2A). The those of the controls. In the MDT group, the patho- diameter of the seminiferous tubules was reduced. Some logical changes at 30 days were much more severe, and spermatogenic cells in the seminiferous tubules were germ cell necrosis was obviously greater than that of the loosened, and the sperm count was obviously reduced. SDT group. Mature sperm were lacking or rarely pres- Quinestrol Induced Testicular Damage 449 ent in the lumen (Fig. 2F). At 60 days, the morphology dant activity by decreasing the antioxidant enzyme activ- and structure of the testes recovered such that it was ity, and at 60 days following treatment, the damage to similar to that of the control (Fig. 2C and 2G). Time- the antioxidant system was alleviated. dependent damage was observed in the testes at 30 days, both in the SDT and MDT groups, and this damage was The Protein concentration in the testis and MDA reversible. concentration in the plasma and testes The MDA and protein concentrations of the different SOD and GSH-Px activities and T-AOC in plasma groups are shown in Table 4. At 15 and 30 days, the At 15 and 30 days after treatment, the SOD and GSH- protein and MDA concentrations in the testes were sig- Px activities and the T-AOC levels of the treated groups nificantly higher than those of the controls. At 15 and were less than those of the control groups (Table 2). At 30 days, the protein concentrations of the treated groups 15 days, quinestrol treatment significantly decreased the were nearly twice as high as that of the control group. SOD (86.5, 95.3, and 99.0 U/ml) and GSH-Px activities At 15 and 30 days, the testicular MDA concentration of (268.6, 303.8, and 365.6 U/ml) and T-AOC (2.13, 3.54, the MDT group was twice as high as that of the control and 6.33 U/ml) both in the MDT and SDT groups com- group while of the SDT group was only 1.7 times than pared with the control groups. At 30 days, antioxidant that of the control. At 60 days, all of these measurements parameters including the SOD activities (84.7, 88.3, and returned to normal levels. At 15, 30, and 60 days, the 100.3 U/ml), GSH-Px activities (234.6, 250.4, and 371.6 change in the MDA levels was not significant in plasma. U/ml), and T-AOC (2.97, 3.22, and 6.67 U/ml) were still The data indicated that the testicular damage was revers- lower both in the MDT and SDT groups compared with ible, although the damage induced by quinestrol was the control groups. At 60 days after treatment, the above significant. parameters were significantly increased. Quinestrol significantly decreased the activity of the antioxidant Discussion enzymes, including SOD and GSH-Px in plasma. But at 60 days after treatment, all of these parameters were In this study, the relationship between reproductive restored. toxicity and oxidative damage induced by quinestrol was studied in male gerbils using daily oral gavage. The SOD and GSH-Px activities and T-AOC in testes results showed that the testes in the MDT group animals The changes in the measured parameters for each were significantly atrophic after 15 and 30 days follow- group in the testes followed the same trend as in the ing quinestrol treatment. Our previous study showed plasma (Table 3). At 15 days, the proportation of the that male gerbils exposed to quinestrol showed a sig- SOD activities to that of the control group in MDT group nificant decrease in epididymides, seminal vesicles, se- is higher than that of the SDT group to control group men quality, and reproductive rates (data not published). (82.6 and 69.6%, respectively), but at 30 days, proporta- The present study shows that the seminiferous tubules tion of the MDT and SDT groups to control group were were severely damaged and degenerated, that a large similar (64.4 and 63.8%). At 15 days, the GSH-Px activ- number of germ cells underwent pyknosis or necrosis ity of the control group was 2.57 and 1.89 times those and that almost no mature sperm were in the lumen. The of the MDT and SDT groups, while at 30 days, the activ- relative testicular weights of the single-dose group were ity of control group was 2.5 times those of both treated significantly lower than those of the control group, but groups. At 15 days, the testicular T-AOC proportions of they were significantly higher than those of the multidose the MDT and SDT groups to the control group were group. Interestingly, the pathological changes were mild similar (76.5 and 82.3%, respectively), but at 30 days, compared with the multidose group, and residual normal the proportions decreaced to 51.3 and 47.3%. At 60 days, tubules could be found. Quinestrol can cause testicular all of the indices were significantly increased in the atrophy, sperm cell death, and testis damage in varying treated gerbils. Quinestrol decreased the total antioxi- degrees depending on the exposure method. In addition, 450 W. Shen, ET AL. b) b) b) b) b) b) , and and , , and , and , and , and c) b) b) , , , , , , b) a) a) CK CK CK , , a) 6.326 ± 1.953 ± 6.326 2.004 ± 0.550 ± 2.004 6.667 ± 2.270 ± 6.667 0.657 ± 2.221 0.719 ± 2.545 6.577 ± 0.806 ± 6.577 12.348 ± 2.342 ± 12.348 13.177 ± 4.093 ± 13.177 13.224 ± 3.426 ± 13.224

c) a) a) d) b) b)

MDT MDT MDT 6.116 ± 0.393 ± 6.116 2.974 ± 0.952 ± 2.974 0.722 ± 1.679 0.325 ± 1.306 1.964 ± 0.649 ± 1.964 2.133 ± 1.975 ± 2.133 13.538 ± 2.300 ± 13.538 10.562 ± 3.240 ± 10.562 12.357 ± 2.960 ± 12.357 T-AOC (U/ml)* T-AOC T-AOC (U/mg prot)* (U/mg T-AOC

a) c) a) a) b) b)

MDA concentration in plasma (nmol/ml)* plasma in concentration MDA SDT SDT SDT 3.542 ± 2.026 ± 3.542 1.686 ± 3.217 0.434 ± 1.827 0.354 ± 1.204 6.502 ± 0.499 ± 6.502 0.434 ± 1.997 11.693 ± 3.185 ± 11.693 13.435 ± 4.728 ± 13.435 13.528 ± 3.359 ± 13.528

b) b) b)

b) b) b)

b) b) b) CK CK CK 1.097 ± 0.218 ± 1.097 1.095 ± 0.124 ± 1.095 0.107 ± 1.141 38.272 ± 10.999 ± 38.272 34.657 ± 4.279 ± 34.657 2.399 ± 32.523 365.627 ± 22.329 ± 365.627 29.421 ± 371.592 356.205 ± 34.375 ± 356.205

c) a) b)

c) a) b) c) c) b)

MDT MDT MDT 2.349 ± 0.127 ± 2.349 2.243 ± 0.172 ± 2.243 1.207 ± 0.214 ± 1.207 GSH-Px (U/ml)* GSH-Px 18.289 ± 9.015 ± 18.289 9.156 ± 12.315 36.212 ± 12.433 ± 36.212 268.571 ± 92.891 ± 268.571 39.452 ± 234.592 367.665 ± 34.748 ± 367.665 GSH-Px (U/mg prot)* (U/mg GSH-Px

a) a) b)

a) a) b) a) a) b)

SDT SDT SDT MDA concentration in testicular tissue (nmol/mg prot)* (nmol/mg tissue testicular in concentration MDA 1.945 ± 0.157 ± 1.945 1.981 ± 0.127 ± 1.981 1.124 ± 0.143 ± 1.124 13.483 ± 5.044 ± 13.483 2.725 ± 12.918 32.913 ± 8.787 ± 32.913 303.781 ± 20.930 ± 303.781 48.343 ± 250.420 372.066 ± 10.991 ± 372.066

b) b) b) b) b) b)

b) b) b)

CK CK CK 1.211 ± 0.088 ± 1.211 1.167 ± 0.104 ± 1.167 1.224 ± 0.091 ± 1.224 192.393 ± 17.682 ± 192.393 100.296 ± 16.040 ± 100.296 29.415 ± 192.369 33.448 ± 196.625 102.716 ± 9.845 ± 102.716 98.967 ± 14.423 ± 98.967

a) a) a) a) b) b)

a) c) b)

MDT MDT MDT SOD (U/ml)* SOD 2.362 ± 0.252 ± 2.362 0.192 ± 2.284 1.124 ± 0.210 ± 1.124 SOD (U/mg prot)* (U/mg SOD 133.923 ± 5.746 ± 133.923 126.551 ± 9.181 ± 126.551 86.534 ± 24.676 ± 86.534 84.657 ± 13.659 ± 84.657 105.539 ± 17.883 ± 105.539 195.123 ± 38.902 ± 195.123

a) c) a) b) b) b)

a) c) b)

SDT SDT SDT Protein concentration in testicular tissue (g/l )* (g/l tissue testicular in concentration Protein 2.261 ± 0.146 ± 2.261 0.241 ± 2.307 1.184 ± 0.149 ± 1.184 88.346 ± 15.587 ± 88.346 95.285 ± 25.789 ± 95.285 Comparison of the SOD and GSH-Px activities, and the T-AOC in plasma at 15, 30, and 60 days after treatment with quinestrol with treatment after days 60 and 30, 15, at plasma in T-AOC the and activities, GSH-Px and SOD the of Comparison quinestrol with treatment after days 60 and 30, 15, at testes the in T-AOC the and activities GSH-Px and SOD, the of Comparison quinestrol with treatment after days 60 and 30, 15, at plasma in concentration MDA the and testes in concentrations MDA and protein the of Comparison 159.388 ± 27.301 ± 159.388 20.562 ± 125.466

100.337 ± 18.396 ± 100.337 42.559 ± 192.646

<0.05). <0.05). <0.05).

P P P

: : : : : : Days 15 30 60 ( significant statistically are row or column same the within letters superscript different bearing values the between differences mean The SD. ± means as expressed are *Results d) Days 15 30 60 *Results are expressed as means ± SD. The mean between the differences values superscript bearing letters different within the same column or row are statistically significant ( c) Days 15 30 60 *Results *Results are expressed as means ± SD. The mean between the differences values bearing superscript letters different within the same column or row are statistically significant ( c) Table 2. Table 3. Table 4. Table Quinestrol Induced Testicular Damage 451 after 60 days following treatment, the absolute and rela- level of lipid peroxidation [12, 18]. Lipid peroxidation tive testicular weights of each group returned to normal (LPO) plays an important role in testicular toxicity and levels, and damage to the testis was recovered, which carcinogenicity. An increase in lipid peroxidation may suggests that damage to the testes of male gerbils in- alter the cellular membrane structure and then block duced by quinestrol is reversible. cellular metabolism [7]. At 15 and 30 days after the The testes were atrophic, their structures were dam- treatment with quinestrol, the increase in MDA concen- aged, and the antioxidant system was also significantly tration in the testes indicated the testicular tissue was changed in the treated groups. The activities of SOD sensitive to quinestrol. The antioxidant enzyme activi- and GSH-Px and the T-AOC were significantly less than ties were decreased, and at the same time, lipid peroxides those of the controls at 15 and 30 days following quin- were generated in the testes, which aggravated sperm estrol treatment (Table 2). The SOD activity was sig- cell damage. The MDA concentration in the single-dose nificantly different between the SDT and MDT groups group was significantly less than that of the multidose after 15 days of treatment. These data indicated that the group (Table 4), which suggested that testicular germ impact of multiple doses on SOD was more severe. In cell damage caused by the multidose treatment was more addition, the protein concentration was increased (Table severe than that of the single-dose treatment. However, 4), so the decrease in the SOD and GSH-Px activities after 60 days, the MDA concentration returned to normal, and the T-AOC were not due to changes in protein con- and the testicular damage was reversed. These data fur- tent. SOD, which catalyses the dismutation of superox- ther confirmed that the level of MDA reflected the degree ide radicals to H2O2 and molecular oxygen [8], is one of of germ cell damage. the most important enzymes for scavenging oxygen free The SOD and GSH-Px activities and the T-AOC in radicals [9]. A decrease in SOD activity that resulted in plasma of the quinestrol-treated gerbils were signifi- ROS generated by testicular tissue that cannot be elimi- cantly decreased compared with those in the control nated over time affects normal germ cell function, lead- (Table 3). However, the MDA concentration was not ing to germ cell death. The germ cell membranes are significantly different compared with the control (Table rich in unsaturated fatty acids that readily undergo lipid 4). This result suggests that other antioxidants partici- peroxidation [6]. GSH-Px scavenges alkyl (R·), RO·, pate in clearing ROS in the plasma, thereby reducing the and ROO· radicals that may be formed from oxidized accumulation of lipid peroxides. Quinestrol mainly in- membrane components, and it uses GSH as a substrate duces reproductive toxicity, while the effect on the rest [1, 2]. Thus, it is possible that the depletion of testicular of the body is weaker. GSH-Px levels by quinestrol may ultimately make the Our data indicate that quinestrol treatment causes an testicular cells even more sensitive to oxidative stress, imbalance in the oxidation and antioxidant system in resulting in damage to the sperm cells. Data from the blood and testicular tissue. Quinestrol induces ROS and T-AOC assay showed that the total antioxidant capacity simultaneously reduces antioxidant enzyme activity, in vivo was significantly decreased in response to quin- leaving germ cells in their oxidation state, which results estrol treatment. This result further confirmed that the in spermatogenic cell damage. Mitochondrial permeabil- testicular damage induced by quinestrol was due to re- ity changes at low concentrations of ROS, which triggers duced levels of antioxidant enzymes. After 60 days fol- the release of cytochrome C and apoptosis factor (AIF) lowing the first treatment, the absolute and relative activated caspase-9 and initiates the apoptosis cascade testicular weights were restored, and the antioxidant [15, 26, 28]. High concentrations of ROS directly dam- enzyme (SOD and GSH-Px) activities and T-AOC re- age the electron transfer chain and reduce the capacity turned to normal. of mitochondrial ATP synthesis significantly [5, 16]. The Lipid peroxidation of unsaturated fatty acids in sperm cells are necrotic because of a lack of energy [5, 16]. membranes is one of the most important effects of ROS- Because ATP is essential for the apoptosis of complex induced cell damage [12]. Malondialdehyde (MDA) is components, the loss of ATP will prolong activation of an end-product of lipid peroxidation that indicates the caspase [27]. Reactive oxygen species also can affect 452 W. Shen, ET AL. biochemical reactions or biological structures, which can cells related to inhibition of cell growth. Cell Biol. Toxicol. in turn affect certain cell signaling cascades. They also 11: 347–354. 8. Fang, Y.Z., Yang, S., and Wu, G. 2002. Free radicals, oxidize thioredoxin and indirect active apoptosis signal antioxidants, and nutrition. Nutrition 18: 872–879. kinase (ASK-1), which leads to apoptosis [11], and in- 9. Jamhan-Arias, A.K., Tyurina, Y.Y., and Kagan, V.E. 2011. duce expression of the transcription factor NF-κB, which Lipid antioxidants: free radical scavenging versus regulation of enzymatic lipid peroxidation. J. Clin. Biochem. Nutr. 48: κ upregulates the NF- B target gene FasL and increases 91–95. apoptotic sensitivity [4, 10]. Additionally, they increase 10. 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