ORIGINAL ARTICLE
Thymoquinone therapy abrogates toxic effect of cadmium on rat testes A. A. Fouad1* & I. Jresat2 1 Pharmacology Division, Department of Biomedical Sciences, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia; 2 Pathology Division, Department of Biomedical Sciences, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia
Keywords Cadmium—rats—testes—thymoquinone Correspondence Amr A. Fouad, Pharmacology Division, Department of Biomedical Sciences, College of Medicine, King Faisal University, Al-Ahsa 31982, Saudi Arabia. Tel.: +96 6501776517; E-mails: [email protected]; [email protected] *Present address: Department of Pharmacology, Faculty of Medicine, Minia University, El-Minia, Egypt. Accepted: February 20, 2014 doi: 10.1111/and.12281
Summary The protective effect of thymoquinone was investigated against cadmiuminduced testicular toxicity in rats. Testicular toxicity was induced by a single intraperitoneal (i.p.) injection of cadmium chloride (2 mg kg1). Thymoquinone treatment (10 mg kg1 day1, i.p.) was applied for five consecutive days, starting 3 days before cadmium administration. Thymoquinone significantly attenuated the cadmium-induced decreases in serum testosterone, and testicular reduced glutathione and superoxide dismutase activity and significantly decreased the elevations of testicular malondialdehyde, nitric oxide and cadmium ion levels resulted from cadmium chloride administration. Also, thymoquinone ameliorated the cadmium-induced testicular tissue injury observed by histopathological examination. In addition, thymoquinone significantly decreased the cadmium-induced expression of inducible nitric oxide synthase, tumour necrosis factor-a, cyclooxygenase-2, nuclear factor-jB and caspase-3 in testicular tissue. It was concluded that thymoquinone, through its antioxidant and anti-inflammatory activities, may represent a potential candidate to protect the testes against the detrimental effect of cadmium exposure.
Introduction The heavy metal cadmium is considered as one of the most common environmental pollutants in the biosphere (Goyer & Clarkson, 2001). It was stated that the provisional tolerable monthly intake for cadmium is 25 lg kg1 body weight, and the safe cadmium levels are 3 lg l1 in drinking water, and an annual average of 5 ng m3 in air (World Health Organization, 2010). However, hazardous exposure to cadmium occurs due to either occupational or nonoccupational reasons. Industrial uses of cadmium in metal plating, pigments, plastics, glass, fertilisers and batteries are the risk factors for occupational exposure. On the other hand, tobacco smoking, air pollution and consumption of cadmium-contaminated drinking water are the major sources for nonoccupational cadmium exposure (Waisberg et al., 2003). Serious injury and dysfunction of different body organs, specially the testes, were reported due to acute and chronic cadmium toxicity in humans and animals (Lauwerys, 1979). Several previous studies demonstrated that cadmium toxicity can lead to testicular tissue injury, © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
decreased testicular weights, compromised testicular function and reduced androgen secretion (Ognjanovic et al., 2010; Yari et al., 2010). Oxidative stress and inflammation are believed to play a major role in the pathogenesis of testicular toxicity and dysfunction induced by cadmium. It is well known that oxidative stress leads to activation of nuclear factor-jB (NF-jB) signalling pathway which is crucial for regulation of many genes involved in inflammatory responses, as tumour necrosis factor-a (TNF-a), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and caspase family of proteases leading eventually to cell death (Tugcu et al., 2010; Chen et al., 2012). Also, previous studies demonstrated the effectiveness of several agents with antioxidant and anti-inflammatory activities against cadmium-induced testicular toxicity (Aktas et al., 2012; Farombi et al., 2012; Fouad & Jresat, 2013). Thymoquinone (TQ; 2-isopropyl-5-methyl-1,4-benzoquinone) is the main active component of Nigella sativa seed oil. TQ possesses prominent antioxidant and anti-inflammatory activities (El-Khouly et al., 2012; Umar et al., 2012; Woo et al., 2012). Previous studies 1
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clearly demonstrated the significant protective effect of TQ in different experimental models of oxidative and inflammatory tissue injuries (Sankaranarayanan & Pari, 2011; Basarslan et al., 2012; Lei et al., 2012; Ullah et al., 2012). A recent study demonstrated that TQ effectively protected against cadmium-induced hepatotoxicity in mice (Zafeer et al., 2012). Also, other recent studies showed that TQ significantly improved semen quality and reproductive characteristics of mice exposed to heat stress (Al-Zahrani et al., 2012), reestablished spermatogenesis after testicular injury due to chronic toluene exposure in rats (Kanter, 2011), protected against methotrexate-induced testicular tissue damage in mice (G€ okcße et al., 2011) and attenuated testicular ischaemia–reperfusion injury resulted from torsion-detorsion in mice (G€ okcße et al., 2010). Therefore, TQ has the potential to protect against testicular damage caused by cadmium exposure, and to the best of our knowledge, this is the first study to investigate the protective effect of TQ against cadmium-induced testicular toxicity. This was encouraging to conduct the present study in order to evaluate the protective effect of TQ in rats exposed to cadmium-induced testicular injury and dysfunction. Also, the possible mechanisms underlying this protective effect were investigated. Materials and methods
tory animals (Institute for Laboratory Animal Research, 2011). Drugs and chemicals Cadmium chloride and TQ powders were purchased from Sigma–Aldrich, St Louis, MO, USA. Cadmium chloride was dissolved in normal saline, while TQ was prepared in 1% aqueous solution of Tween-80. The doses of cadmium chloride and TQ used in the present study were selected based on our preliminary experiments and in accordance with previous reports (Gupta et al., 2004; G€ okcße et al., 2010, 2011). Experimental protocol The rats were randomly divided into four groups (n = 8, each). The first (control) group received a single intraperitoneal (i.p.) injection of normal saline (vehicle of cadmium chloride). Testicular toxicity was induced in rats of the second and third groups by a single injection of cadmium chloride (2 mg kg1, i.p.). The animals of the second and third groups received the vehicle of TQ (1% aqueous solution of Tween-80) or TQ (10 mg kg day1, i.p.), respectively, for five consecutive days starting 3 days before cadmium chloride administration. The fourth group of animals received TQ for five consecutive days without induction of cadmium toxicity.
Animals
Sampling and biochemical analyses
Male Sprague–Dawley rats, weighing 250 10 g, were obtained from the Animal House, College of Medicine, King Faisal University. The animals were kept at standard housing facilities (24 1 °C, 45 5% humidity and 12 h light–12 h dark cycle). They were supplied with standard laboratory chow and water ad libitum and left to acclimatise for 1 week before the experiments. The experimental procedures were carried out in accordance with international guidelines for care and use of labora-
The rats were euthanised 48 h following cadmium chloride administration. Blood samples were collected through a puncture in the left ventricle, left 60 min to clot and centrifuged at 2430 g for 10 min. The obtained clear sera were stored at 80 °C, and subsequently serum testosterone level was measured using rat testosterone enzyme-linked immunosorbent assay (ELISA) kit following the instructions of the manufacturer (DRG Diagnostics, GmbH, Marburg, Germany).
Table 1 Effects of thymoquinone (TQ) treatment on serum testosterone level, and testicular malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO) and cadmium ion levels, and superoxide dismutase (SOD) activity in rats exposed to cadmium (Cd) toxicity Vehicle + Cd
Control Serum testosterone (ng ml1) MDA (nmol g1 tissue) GSH (mmol g1 tissue) NO (nmol per 100 mg tissue) SOD (U mg1 protein) Cadmium ion (lg g1 tissue)
2.01 11.25 2.63 30.81 58.18 1.26
0.24 1.87 0.15 4.45 4.50 0.14
0.63 127.94 0.81 88.52 23.61 5.59
0.07* 11.37* 0.09* 7.17* 2.19* 0.62*
TQ + Cd 1.44 28.17 3.01 46.92 40.36 1.81
TQ 0.17** 3.10** 0.57** 2.61** 2.62*,** 0.08*
2.25 14.86 2.85 21.74 51.37 1.42
0.13 2.61 0.11 2.53 3.95 0.10
All the values are expressed as mean SEM, n = 8 in each group. *P < 0.05 versus control group. **P < 0.05 versus vehicle + cadmium group.
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Fig. 1 Photomicrographs of rat testes (H&E) from (a, 2009) control group showing normal architecture of testicular tissue with normal spermatogenesis in seminiferous tubules; (b, 2009 and c, 4009) cadmium group without thymoquinone treatment showing widespread necrosis, vacuolar degeneration of seminiferous tubular cells (c, black arrows), marked reduction in spermatogenesis, interstitial tissue oedema (b, white arrows) and haemorrhages (c, arrow heads); (d, 2009) Thymoquinone plus cadmium group showing a histological picture comparable to that of the control group with preservation of normal spermatogenesis in most of seminiferous tubules; (e) Johnsen scoring for the level of spermatogenesis in the different groups, data are mean SEM of eight rats, *P < 0.05 versus control group, •P < 0.05 versus cadmium group without thymoquinone treatment. Cd, cadmium; TQ, thymoquinone.
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(d) Fig. 2 Immunohistochemical staining of inducible nitric oxide synthase (iNOS) in rat testes from: (a, 2009) control group showing no expression of iNOS; (b, 2009 and inserted figure, 4009) cadmium group without thymoquinone (TQ) treatment showing a significant increase in iNOS immunoreactivity in the cytoplasm of seminiferous tubular cells; (c, 2009) TQ plus cadmium group showing a significant decrease in iNOS immunostaining. Brown colour indicates iNOS positivity; (d) percentage expression of iNOS, data are mean SEM of eight rats, *P < 0.05 versus control group, •P < 0.05 versus cadmium group without TQ treatment. ND, nondetectable; Cd, cadmium.
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The testes were removed and dissected free from the surrounding fat and connective tissue. The right testis obtained from each animal was homogenised in cold potassium phosphate buffer (0.05 M, pH 7.4). The testicular homogenates were centrifuged at 2430 g for 10 min at 4 °C, and the resulting supernatant was kept at 80 °C. Subsequently, the supernatant of testicular homogenates was used for measurement of malondialdehyde (MDA), as an indicator of lipid peroxidation, and reduced glutathione (GSH) levels, and superoxide dismutase (SOD) activity according to the recommendations of the manufacturer (Biodiagnostic, Cairo, Egypt). The testicular level of nitric oxide (NO) was assayed using colorimetric assay kit following the manufacturer’s instructions (Cayman Chemical Company, Ann Arbor, MI, USA). Also, parts of the testicular tissue were dried overnight at 80 °C and the dry weight was recorded. The samples were then digested with equal volumes of 30% (w/v) H2O2 and 70% (w/v) nitric acid, and the clear digest was diluted with ultrapure water (1 : 3). Testicular cadmium ion level was measured using inductively coupled plasma
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Fig. 3 Immunohistochemical staining of tumour necrosis factor-a (TNF-a) in rat testes from: (a, 2009) control group showing no expression of TNF-a; (b, 2009 and inserted figure, 4009) cadmium group without thymoquinone (TQ) treatment showing a significant increase in TNF-a immunoreactivity in the cytoplasm of seminiferous tubular cells; (c, 2009) TQ plus cadmium group showing a significant reduction in TNF-a immunostaining. Brown colour indicates TNF-a positivity; (d) percentage expression of TNF-a, data are mean SEM of eight rats, *P < 0.05 versus control group, •P < 0.05 versus cadmium group without TQ treatment. ND, nondetectable; Cd, cadmium.
optical emission spectrometer (Optima 4300 DV; PerkinElmer, Shelton, CT, USA) at 228.8 nm, with sample-based standard. Histopathological examination The left testis obtained from each animal was fixed in Bouin’s solution, dehydrated in ascending grades of ethanol and embedded in paraffin. Sections at 4 lm thickness were taken, stained with haematoxylin and eosin (H&E) and examined under light microscope by a pathologist unaware of the treatment protocol. In addition, the level of spermatogenesis in the testicular tissue was assessed using Johnsen score (Johnsen, 1970). A scale from 1 to 10 was given to 100 tubules per slide as follows: 10 = full spermatogenesis, 9 = slightly impaired spermatogenesis, 8 = few spermatozoa, 7 = no spermatozoa but many spermatids, 6 = few spermatids, 5 = no spematids but many spermatocytes, 4 = few spermatocytes, 3 = spermatogonia only, 2 = no germ cells but only Sertoli cells, 1 = atrophic tubules without seminiferous epithelial cells.
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(d) Fig. 4 Immunohistochemical staining of cyclooxygenase-2 (COX-2) in rat testes from: (a, 2009) control group showing no expression of COX-2; (b, 2009 and inserted figure, 4009) cadmium group without thymoquinone (TQ) treatment showing a significant increase in COX-2 immunoreactivity in the cytoplasm of seminiferous tubular cells; (c, 2009) TQ plus cadmium group showing a significant decrease in COX-2 immunostaining. Brown colour indicates COX-2 positivity; (d) percentage expression of COX-2, data are mean SEM of eight rats, *P < 0.05 versus control group, •P < 0.05 versus cadmium group without TQ treatment. ND, nondetectable; Cd, cadmium.
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Immunohistochemical examinations Four-micrometre-thick sections prepared from different animal groups were deparaffinised and rehydrated, and endogenous peroxidase activity was blocked with 3% H2O2 in methanol. Sections were pre-treated in citrate buffer (pH 6.0) in a microwave. Sections were incubated with rabbit polyclonal antibodies specific for the rat targets. The antibodies used were anti-iNOS, anti-COX-2, anti-NF-jB and anticaspase-3 antibodies (Dilution 1 : 1000; Thermo Scientific, Fremont, CA, USA) and anti-TNF-a (Dilution 1 : 500; US Biological, Swampscott, MA, USA). The sections were incubated with biotinylated goat antipolyvalent, then with streptavidin peroxidase and finally with diaminobenzidine plus chromogen. Slides were counterstained with haematoxylin. The slides were visualised under light microscope, and the extent of cell immunopositivity was assessed. The number of immunopositive cells was counted in five separate microscopic fields in each slide, the mean number for each slide was obtained, and then the mean SEM was calculated for each group (eight slides). The same procedures were repeated using normal © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
rabbit serum instead of the primary antibodies to obtain negative control and indicate the specificity of the used antibodies (Rasmussen, 2009).
Statistical analysis The values are expressed as mean SEM. The results were analysed by one-way analysis of variance (ANOVA) followed by Tukey’s test for post hoc comparisons using SPSS for Windows (version 18, IBM Corporation, Armonk, NY, USA). Differences were considered significant at P < 0.05. Results Biochemical analyses Cadmium chloride administration resulted in significant decreases in serum testosterone level, testicular GSH and SOD activity, and significant increases in testicular MDA, NO and cadmium ion levels as compared to the control group (Table 1). Rats treated with TQ showed significantly 5
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(d) Fig. 5 Immunohistochemical staining of nuclear factor-jB (NF-jB) in rat testes from: (a, 2009) control showing no expression of NF-jB; (b, 2009 and inserted figure, 4009) cadmium group without thymoquinone (TQ) treatment showing a significant increase in NF-jB immunoreactivity in the cytoplasm of seminiferous tubular cells; (c, 2009) TQ plus cadmium group demonstrating a significant reduction in NF-jB immunostaining. Brown colour indicates NF-jB positivity; (d) percentage expression of NF-jB, data are mean SEM of eight rats, *P < 0.05 versus control group, •P < 0.05 versus cadmium group without TQ treatment. ND, nondetectable; Cd, cadmium.
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higher serum testosterone level, and testicular GSH and SOD activity, and significantly lower levels of MDA, NO and cadmium ion in testicular homogenates as compared to the cadmium group nontreated with TQ (Table 1). Testicular histopathology Cadmium administration caused widespread necrosis and vacuolisation of the seminiferous tubular cells, associated with impaired spermatogenesis, interstitial tissue oedema and haemorrhages. However, TQ treatment markedly ameliorated the cadmium-induced damage of testicular tissue and preserved spermatogenesis in most of seminiferous tubules (Fig. 1). Also, Johnsen score was significantly decreased in cadmium group nontreated with TQ as compared to the control group. On the other hand, TQ treatment resulted in a significant increase in Johnsen score (Fig. 1). Testicular immunohistochemistry Significant increases in the expression of iNOS, TNF-a, COX-2, NF-jB and caspase-3 in the cells of seminiferous 6
tubules were observed in rats received cadmium chloride as compared to the control animals. On the other hand, TQ treatment significantly decreased the cadmiuminduced expression of iNOS, TNF-a, COX-2, NF-jB and caspase-3 in the cells of seminiferous tubules as compared to the cadmium group nontreated with TQ (Figs 2–6). The slides from the cadmium group without TQ treatment which were incubated with normal rabbit serum instead of the primary antibodies showed no staining at all indicating the specificity of the used antibodies (figures not shown). Discussion The present work, in agreement with previous studies, confirmed that oxidative stress, increased lipid peroxidation, depletion of antioxidant defences and increased production of pro-inflammatory mediators are implicated in the pathogenesis of cadmium-induced testicular toxicity (Aktas et al., 2012; Farombi et al., 2012; Fouad & Jresat, 2013). Also, the present study demonstrated that TQ treatment provided a significant protective effect against testicular injury caused by cadmium in rats as indicated by the improvement in the disturbed biochemical parameters and © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
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(d) Fig. 6 Immunohistochemical staining of caspase-3 in rat testes from: (a, 2009) control group showing no expression of caspase-3; (b, 2009 and inserted figure, 4009) cadmium group without thymoquinone (TQ) treatment showing a significant increase in caspase-3 immunoreactivity in the cytoplasm of seminiferous tubular cells; (c, 2009) TQ plus cadmium group demonstrating a significant reduction in caspase-3 immunostaining. Brown colour indicates caspase-3 positivity; (d) percentage expression of caspase-3, data are mean SEM of eight rats, *P < 0.05 versus control group, •P < 0.05 versus cadmium group without TQ treatment. ND, nondetectable; Cd, cadmium.
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amelioration of testicular tissue damage observed by histopathological and immunohistochemical examinations. Recent evidence suggests that cadmium-induced testicular toxicity occurs as a result of oxidative stress and increased generation of reactive oxygen species (Ji et al., 2012; Zhang et al., 2012). Increased production of reactive oxygen species depletes reduced glutathione and enhances lipid peroxidation in testicular tissue (Abarikwu et al., 2013; Spiazzi et al., 2013). Also, cadmium induces a cascade of inflammatory reactions with increased production of pro-inflammatory cytokines, particularly TNFa which is responsible for further testicular tissue injury (Al-Azemi et al., 2010; De Freitas et al., 2012). In addition, increased NO production in the testes is involved in the pathogenesis of cadmium toxicity (Gunnarsson et al., 2003; Fouad et al., 2009). It was reported that inflammatory stimuli, particularly TNF-a, can increase the expression of iNOS which catalyses the production of a large amount of NO. Excess NO reacts with superoxide anion to generate peroxynitrite radical responsible for cell damage by nitrating cellular macromolecules (Kobayashi, 2010). Also, excess NO depletes intracellular GSH and increases the susceptibility to oxidative stress (Ekici et al., © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
2012). Moreover, cadmium induces the expression of COX-2, which increases the production of inflammatory prostaglandins implicated in testicular tissue injury (Gunnarsson et al., 2003; Fouad et al., 2013). Thymoquinone is the main active constituent isolated from Nigella sativa seed oil and is known to have multiple biological effects. TQ exerts marked antioxidant activity, scavenges reactive oxygen radicals, suppresses lipid peroxidation and maintains the antioxidant defence mechanisms during free radical reactions (El-Khouly et al., 2012; Umar et al., 2012; Woo et al., 2012). Also, TQ inhibits iNOS activity and decreases NO production and therefore prevents nitrosative tissue stress (Ammar et al., 2011; Kanter, 2011). In addition, TQ exhibits significant anti-inflammatory activity because it reduces the release of pro-inflammatory cytokines (Vaillancourt et al., 2011; Umar et al., 2012) and inhibits COX-2 which is capable of producing large amounts of inflammatory prostaglandins (El Mezayen et al., 2006; Vaillancourt et al., 2011). This in accordance with the present results which revealed that TQ treatment significantly suppressed lipid peroxidation, prevented the depletion of GSH, decreased NO production and reduced the expression of iNOS, 7
Thymoquinone prevents cadmium testicular injury
TNF-a and COX-2 in the testes of rats exposed to cadmium toxicity. It was also reported that cadmium caused NF-jB activation with subsequent inflammatory reactions responsible for testicular tissue injury (Fouad & Jresat, 2013; Fouad et al., 2013). Elevated TNF-a is a well-known inducer of NF-jB signalling pathway (Li & Verma, 2002). The present results revealed that TQ significantly reduced the expression of testicular NF-jB in rats exposed to cadmium toxicity. This is in accordance with previous results which demonstrated that TQ provided a significant anti-inflammatory effect by inhibiting the activity of NF-jB (Chehl et al., 2009; Vaillancourt et al., 2011). Therefore, the testicular protective effect of TQ can be attributed to its ability to inhibit NF-jB signalling pathway which promotes the transcription of TNF-a, COX-2 and iNOS genes (Abd ElGhany et al., 2009; Chehl et al., 2009; Shen et al., 2013). The present study revealed that TQ significantly decreased the cadmium-induced expression of caspase-3, an executioner of cell apoptosis, in the testicular tissue. This is in agreement with previous studies which showed that TQ provided a significant anti-apoptotic effect by inhibiting caspase-3 activity (Helal, 2010; Ullah et al., 2012). Therefore, it could be stated that TQ protected against cadmiuminduced testicular cell apoptosis. The reduced caspase-3 activity observed with TQ treatment may be due to its free radical scavenging activity and anti-inflammatory action with reduced expression of TNF-a and NF-jB. Also, to the best of our knowledge, this is the first study to demonstrate the inhibitory effect of TQ on the expression of NF-jB, COX-2, TNF-a and caspase-3 in the testicular tissue. The present work revealed that TQ significantly attenuated the increase in testicular cadmium ion concentration resulted from cadmium chloride administration. This may be due to the ability of TQ to prevent cadmium-induced depletion of GSH which has antioxidant and metal-chelating activities (Rooney, 2007). The thiol groups in GSH can bind heavy metals with high affinity and play an important role in intracellular heavy metal detoxification (Satoh et al., 2000). It was concluded that TQ significantly abrogated cadmium-induced testicular injury and dysfunction in rats. The antioxidant and anti-inflammatory activities of TQ can be considered the main factors responsible for the testicular protective effect. Therefore, TQ may represent a potential therapeutic option to protect against the deleterious effect of cadmium exposure on the testes. References Abarikwu SO, Iserhienrhien BO, Badejo TA (2013) Rutin- and selenium-attenuated cadmium-induced testicular pathophysiology in rats. Hum Exp Toxicol 32:395–406.
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sodium-induced colitis in mice. Dig Dis Sci 57:2296– 2303. Li Q, Verma IM (2002) NF-kappaB regulation in immune system. Nat Rev Immunol 2:725–734. Ognjanovic BI, Markovic SD, Ethordevic NZ, Trbojevic IS, Stajn AS, Saicic ZS (2010) Cadmium-induced lipid peroxidation and changes in antioxidant defense system in the rat testes: protective role of coenzyme Q(10) and vitamin E. Reprod Toxicol 29:191–197. Rasmussen OF (2009) Controls. In: Immunohistochemical Staining Methods. Kumar GL, Rudbeck L (eds.). Dako North America, Carpinteria, CA, p 127. Rooney JP (2007) The role of thiols, dithiols, nutritional factors and interacting ligands in the toxicology of mercury. Toxicology 234:145–156. Sankaranarayanan C, Pari L (2011) Thymoquinone ameliorates chemical induced oxidative stress and b-cell damage in experimental hyperglycemic rats. Chem Biol Interact 190:148–154. Satoh M, Shimada A, Zhang B, Tohyama C (2000) Renal toxicity caused by cisplatinum in glutathione-depleted metallothionein-null mice. Biochem Pharmacol 60:1729– 1734. Shen YC, Yen JC, Liou KT (2013) Ameliorative effects of caffeic acid phenethyl ester on an eccentric exercise-induced skeletal muscle injury by down-regulating NF-jB mediated inflammation. Pharmacology 91:219–228. Spiazzi CC, Manfredini V, Barcellos da Silva FE, Flores EM, Izaguirry AP, Vargas LM, Soares MB, Santos FW (2013) c-Oryzanol protects against acute cadmium-induced oxidative damage in mice testes. Food Chem Toxicol 55:526–532. Tugcu V, Gedikbasßi A, Mutlu B, G€ uner E, Uhri M, Andican G, Ozbek E, Tasßßci AI (2010) Increased testicular 8-hydroxy20 -deoxyguanosine (8-OHdG) and inducible nitric oxide synthetase (iNOS) and nuclear factor jB (NF-jB) expressions in experimental rat varicocele. Arch Ital Urol Androl 82:148–153. Ullah I, Ullah N, Naseer MI, Lee HY, Kim MO (2012) Neuroprotection with metformin and thymoquinone against ethanol-induced apoptotic neurodegeneration in prenatal rat cortical neurons. BMC Neurosci 13:11. Umar S, Zargan J, Umar K, Ahmad S, Katiyar CK, Khan HA (2012) Modulation of the oxidative stress and inflammatory cytokine response by thymoquinone in the collagen induced arthritis in Wistar rats. Chem Biol Interact 197:40–46. Vaillancourt F, Silva P, Shi Q, Fahmi H, Fernandes JC, Benderdour M (2011) Elucidation of molecular mechanisms underlying the protective effects of thymoquinone against rheumatoid arthritis. J Cell Biochem 112:107–117. Waisberg M, Joseph P, Hale B, Beyersmann D (2003) Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 192:95–117.
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Woo CC, Kumar AP, Sethi G, Tan KHB (2012) Thymoquinone: potential cure for inflammatory disorders and cancer. Biochem Pharmacol 83:443–451. World Health Organization (2010) Exposure to cadmium: a major public health concern. Geneva, Switzerland. Available at: http://www.who.int/ipcs/features/cadmium. Yari A, Asadi MH, Bahadoran H, Dashtnavard H, Imani H, Naghii MR (2010) Cadmium toxicity in spermatogenesis
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and protective effects of L-carnitine in adult male rats. Biol Trace Elem Res 137:216–225. Zafeer MF, Waseem M, Chaudhary S, Parvez S (2012) Cadmium-induced hepatotoxicity and its abrogation by thymoquinone. J Biochem Mol Toxicol 26:199–205. Zhang H, Cai C, Shi C, Cao H, Han Z, Jia X (2012) Cadmiuminduced oxidative stress and apoptosis in the testes of frog Rana limnocharis. Aquat Toxicol 122–123:67–74.
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Thymoquinone therapy abrogates toxic effect of cadmium on rat testes A. A. Fouad1* & I. Jresat2 1 Pharmacology Division, Department of Biomedical Sciences, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia; 2 Pathology Division, Department of Biomedical Sciences, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia
Keywords Cadmium—rats—testes—thymoquinone Correspondence Amr A. Fouad, Pharmacology Division, Department of Biomedical Sciences, College of Medicine, King Faisal University, Al-Ahsa 31982, Saudi Arabia. Tel.: +96 6501776517; E-mails: [email protected]; [email protected] *Present address: Department of Pharmacology, Faculty of Medicine, Minia University, El-Minia, Egypt. Accepted: February 20, 2014 doi: 10.1111/and.12281
Summary The protective effect of thymoquinone was investigated against cadmiuminduced testicular toxicity in rats. Testicular toxicity was induced by a single intraperitoneal (i.p.) injection of cadmium chloride (2 mg kg1). Thymoquinone treatment (10 mg kg1 day1, i.p.) was applied for five consecutive days, starting 3 days before cadmium administration. Thymoquinone significantly attenuated the cadmium-induced decreases in serum testosterone, and testicular reduced glutathione and superoxide dismutase activity and significantly decreased the elevations of testicular malondialdehyde, nitric oxide and cadmium ion levels resulted from cadmium chloride administration. Also, thymoquinone ameliorated the cadmium-induced testicular tissue injury observed by histopathological examination. In addition, thymoquinone significantly decreased the cadmium-induced expression of inducible nitric oxide synthase, tumour necrosis factor-a, cyclooxygenase-2, nuclear factor-jB and caspase-3 in testicular tissue. It was concluded that thymoquinone, through its antioxidant and anti-inflammatory activities, may represent a potential candidate to protect the testes against the detrimental effect of cadmium exposure.
Introduction The heavy metal cadmium is considered as one of the most common environmental pollutants in the biosphere (Goyer & Clarkson, 2001). It was stated that the provisional tolerable monthly intake for cadmium is 25 lg kg1 body weight, and the safe cadmium levels are 3 lg l1 in drinking water, and an annual average of 5 ng m3 in air (World Health Organization, 2010). However, hazardous exposure to cadmium occurs due to either occupational or nonoccupational reasons. Industrial uses of cadmium in metal plating, pigments, plastics, glass, fertilisers and batteries are the risk factors for occupational exposure. On the other hand, tobacco smoking, air pollution and consumption of cadmium-contaminated drinking water are the major sources for nonoccupational cadmium exposure (Waisberg et al., 2003). Serious injury and dysfunction of different body organs, specially the testes, were reported due to acute and chronic cadmium toxicity in humans and animals (Lauwerys, 1979). Several previous studies demonstrated that cadmium toxicity can lead to testicular tissue injury, © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
decreased testicular weights, compromised testicular function and reduced androgen secretion (Ognjanovic et al., 2010; Yari et al., 2010). Oxidative stress and inflammation are believed to play a major role in the pathogenesis of testicular toxicity and dysfunction induced by cadmium. It is well known that oxidative stress leads to activation of nuclear factor-jB (NF-jB) signalling pathway which is crucial for regulation of many genes involved in inflammatory responses, as tumour necrosis factor-a (TNF-a), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and caspase family of proteases leading eventually to cell death (Tugcu et al., 2010; Chen et al., 2012). Also, previous studies demonstrated the effectiveness of several agents with antioxidant and anti-inflammatory activities against cadmium-induced testicular toxicity (Aktas et al., 2012; Farombi et al., 2012; Fouad & Jresat, 2013). Thymoquinone (TQ; 2-isopropyl-5-methyl-1,4-benzoquinone) is the main active component of Nigella sativa seed oil. TQ possesses prominent antioxidant and anti-inflammatory activities (El-Khouly et al., 2012; Umar et al., 2012; Woo et al., 2012). Previous studies 1
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clearly demonstrated the significant protective effect of TQ in different experimental models of oxidative and inflammatory tissue injuries (Sankaranarayanan & Pari, 2011; Basarslan et al., 2012; Lei et al., 2012; Ullah et al., 2012). A recent study demonstrated that TQ effectively protected against cadmium-induced hepatotoxicity in mice (Zafeer et al., 2012). Also, other recent studies showed that TQ significantly improved semen quality and reproductive characteristics of mice exposed to heat stress (Al-Zahrani et al., 2012), reestablished spermatogenesis after testicular injury due to chronic toluene exposure in rats (Kanter, 2011), protected against methotrexate-induced testicular tissue damage in mice (G€ okcße et al., 2011) and attenuated testicular ischaemia–reperfusion injury resulted from torsion-detorsion in mice (G€ okcße et al., 2010). Therefore, TQ has the potential to protect against testicular damage caused by cadmium exposure, and to the best of our knowledge, this is the first study to investigate the protective effect of TQ against cadmium-induced testicular toxicity. This was encouraging to conduct the present study in order to evaluate the protective effect of TQ in rats exposed to cadmium-induced testicular injury and dysfunction. Also, the possible mechanisms underlying this protective effect were investigated. Materials and methods
tory animals (Institute for Laboratory Animal Research, 2011). Drugs and chemicals Cadmium chloride and TQ powders were purchased from Sigma–Aldrich, St Louis, MO, USA. Cadmium chloride was dissolved in normal saline, while TQ was prepared in 1% aqueous solution of Tween-80. The doses of cadmium chloride and TQ used in the present study were selected based on our preliminary experiments and in accordance with previous reports (Gupta et al., 2004; G€ okcße et al., 2010, 2011). Experimental protocol The rats were randomly divided into four groups (n = 8, each). The first (control) group received a single intraperitoneal (i.p.) injection of normal saline (vehicle of cadmium chloride). Testicular toxicity was induced in rats of the second and third groups by a single injection of cadmium chloride (2 mg kg1, i.p.). The animals of the second and third groups received the vehicle of TQ (1% aqueous solution of Tween-80) or TQ (10 mg kg day1, i.p.), respectively, for five consecutive days starting 3 days before cadmium chloride administration. The fourth group of animals received TQ for five consecutive days without induction of cadmium toxicity.
Animals
Sampling and biochemical analyses
Male Sprague–Dawley rats, weighing 250 10 g, were obtained from the Animal House, College of Medicine, King Faisal University. The animals were kept at standard housing facilities (24 1 °C, 45 5% humidity and 12 h light–12 h dark cycle). They were supplied with standard laboratory chow and water ad libitum and left to acclimatise for 1 week before the experiments. The experimental procedures were carried out in accordance with international guidelines for care and use of labora-
The rats were euthanised 48 h following cadmium chloride administration. Blood samples were collected through a puncture in the left ventricle, left 60 min to clot and centrifuged at 2430 g for 10 min. The obtained clear sera were stored at 80 °C, and subsequently serum testosterone level was measured using rat testosterone enzyme-linked immunosorbent assay (ELISA) kit following the instructions of the manufacturer (DRG Diagnostics, GmbH, Marburg, Germany).
Table 1 Effects of thymoquinone (TQ) treatment on serum testosterone level, and testicular malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO) and cadmium ion levels, and superoxide dismutase (SOD) activity in rats exposed to cadmium (Cd) toxicity Vehicle + Cd
Control Serum testosterone (ng ml1) MDA (nmol g1 tissue) GSH (mmol g1 tissue) NO (nmol per 100 mg tissue) SOD (U mg1 protein) Cadmium ion (lg g1 tissue)
2.01 11.25 2.63 30.81 58.18 1.26
0.24 1.87 0.15 4.45 4.50 0.14
0.63 127.94 0.81 88.52 23.61 5.59
0.07* 11.37* 0.09* 7.17* 2.19* 0.62*
TQ + Cd 1.44 28.17 3.01 46.92 40.36 1.81
TQ 0.17** 3.10** 0.57** 2.61** 2.62*,** 0.08*
2.25 14.86 2.85 21.74 51.37 1.42
0.13 2.61 0.11 2.53 3.95 0.10
All the values are expressed as mean SEM, n = 8 in each group. *P < 0.05 versus control group. **P < 0.05 versus vehicle + cadmium group.
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Fig. 1 Photomicrographs of rat testes (H&E) from (a, 2009) control group showing normal architecture of testicular tissue with normal spermatogenesis in seminiferous tubules; (b, 2009 and c, 4009) cadmium group without thymoquinone treatment showing widespread necrosis, vacuolar degeneration of seminiferous tubular cells (c, black arrows), marked reduction in spermatogenesis, interstitial tissue oedema (b, white arrows) and haemorrhages (c, arrow heads); (d, 2009) Thymoquinone plus cadmium group showing a histological picture comparable to that of the control group with preservation of normal spermatogenesis in most of seminiferous tubules; (e) Johnsen scoring for the level of spermatogenesis in the different groups, data are mean SEM of eight rats, *P < 0.05 versus control group, •P < 0.05 versus cadmium group without thymoquinone treatment. Cd, cadmium; TQ, thymoquinone.
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(d) Fig. 2 Immunohistochemical staining of inducible nitric oxide synthase (iNOS) in rat testes from: (a, 2009) control group showing no expression of iNOS; (b, 2009 and inserted figure, 4009) cadmium group without thymoquinone (TQ) treatment showing a significant increase in iNOS immunoreactivity in the cytoplasm of seminiferous tubular cells; (c, 2009) TQ plus cadmium group showing a significant decrease in iNOS immunostaining. Brown colour indicates iNOS positivity; (d) percentage expression of iNOS, data are mean SEM of eight rats, *P < 0.05 versus control group, •P < 0.05 versus cadmium group without TQ treatment. ND, nondetectable; Cd, cadmium.
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The testes were removed and dissected free from the surrounding fat and connective tissue. The right testis obtained from each animal was homogenised in cold potassium phosphate buffer (0.05 M, pH 7.4). The testicular homogenates were centrifuged at 2430 g for 10 min at 4 °C, and the resulting supernatant was kept at 80 °C. Subsequently, the supernatant of testicular homogenates was used for measurement of malondialdehyde (MDA), as an indicator of lipid peroxidation, and reduced glutathione (GSH) levels, and superoxide dismutase (SOD) activity according to the recommendations of the manufacturer (Biodiagnostic, Cairo, Egypt). The testicular level of nitric oxide (NO) was assayed using colorimetric assay kit following the manufacturer’s instructions (Cayman Chemical Company, Ann Arbor, MI, USA). Also, parts of the testicular tissue were dried overnight at 80 °C and the dry weight was recorded. The samples were then digested with equal volumes of 30% (w/v) H2O2 and 70% (w/v) nitric acid, and the clear digest was diluted with ultrapure water (1 : 3). Testicular cadmium ion level was measured using inductively coupled plasma
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Fig. 3 Immunohistochemical staining of tumour necrosis factor-a (TNF-a) in rat testes from: (a, 2009) control group showing no expression of TNF-a; (b, 2009 and inserted figure, 4009) cadmium group without thymoquinone (TQ) treatment showing a significant increase in TNF-a immunoreactivity in the cytoplasm of seminiferous tubular cells; (c, 2009) TQ plus cadmium group showing a significant reduction in TNF-a immunostaining. Brown colour indicates TNF-a positivity; (d) percentage expression of TNF-a, data are mean SEM of eight rats, *P < 0.05 versus control group, •P < 0.05 versus cadmium group without TQ treatment. ND, nondetectable; Cd, cadmium.
optical emission spectrometer (Optima 4300 DV; PerkinElmer, Shelton, CT, USA) at 228.8 nm, with sample-based standard. Histopathological examination The left testis obtained from each animal was fixed in Bouin’s solution, dehydrated in ascending grades of ethanol and embedded in paraffin. Sections at 4 lm thickness were taken, stained with haematoxylin and eosin (H&E) and examined under light microscope by a pathologist unaware of the treatment protocol. In addition, the level of spermatogenesis in the testicular tissue was assessed using Johnsen score (Johnsen, 1970). A scale from 1 to 10 was given to 100 tubules per slide as follows: 10 = full spermatogenesis, 9 = slightly impaired spermatogenesis, 8 = few spermatozoa, 7 = no spermatozoa but many spermatids, 6 = few spermatids, 5 = no spematids but many spermatocytes, 4 = few spermatocytes, 3 = spermatogonia only, 2 = no germ cells but only Sertoli cells, 1 = atrophic tubules without seminiferous epithelial cells.
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(d) Fig. 4 Immunohistochemical staining of cyclooxygenase-2 (COX-2) in rat testes from: (a, 2009) control group showing no expression of COX-2; (b, 2009 and inserted figure, 4009) cadmium group without thymoquinone (TQ) treatment showing a significant increase in COX-2 immunoreactivity in the cytoplasm of seminiferous tubular cells; (c, 2009) TQ plus cadmium group showing a significant decrease in COX-2 immunostaining. Brown colour indicates COX-2 positivity; (d) percentage expression of COX-2, data are mean SEM of eight rats, *P < 0.05 versus control group, •P < 0.05 versus cadmium group without TQ treatment. ND, nondetectable; Cd, cadmium.
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Immunohistochemical examinations Four-micrometre-thick sections prepared from different animal groups were deparaffinised and rehydrated, and endogenous peroxidase activity was blocked with 3% H2O2 in methanol. Sections were pre-treated in citrate buffer (pH 6.0) in a microwave. Sections were incubated with rabbit polyclonal antibodies specific for the rat targets. The antibodies used were anti-iNOS, anti-COX-2, anti-NF-jB and anticaspase-3 antibodies (Dilution 1 : 1000; Thermo Scientific, Fremont, CA, USA) and anti-TNF-a (Dilution 1 : 500; US Biological, Swampscott, MA, USA). The sections were incubated with biotinylated goat antipolyvalent, then with streptavidin peroxidase and finally with diaminobenzidine plus chromogen. Slides were counterstained with haematoxylin. The slides were visualised under light microscope, and the extent of cell immunopositivity was assessed. The number of immunopositive cells was counted in five separate microscopic fields in each slide, the mean number for each slide was obtained, and then the mean SEM was calculated for each group (eight slides). The same procedures were repeated using normal © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
rabbit serum instead of the primary antibodies to obtain negative control and indicate the specificity of the used antibodies (Rasmussen, 2009).
Statistical analysis The values are expressed as mean SEM. The results were analysed by one-way analysis of variance (ANOVA) followed by Tukey’s test for post hoc comparisons using SPSS for Windows (version 18, IBM Corporation, Armonk, NY, USA). Differences were considered significant at P < 0.05. Results Biochemical analyses Cadmium chloride administration resulted in significant decreases in serum testosterone level, testicular GSH and SOD activity, and significant increases in testicular MDA, NO and cadmium ion levels as compared to the control group (Table 1). Rats treated with TQ showed significantly 5
Thymoquinone prevents cadmium testicular injury
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(d) Fig. 5 Immunohistochemical staining of nuclear factor-jB (NF-jB) in rat testes from: (a, 2009) control showing no expression of NF-jB; (b, 2009 and inserted figure, 4009) cadmium group without thymoquinone (TQ) treatment showing a significant increase in NF-jB immunoreactivity in the cytoplasm of seminiferous tubular cells; (c, 2009) TQ plus cadmium group demonstrating a significant reduction in NF-jB immunostaining. Brown colour indicates NF-jB positivity; (d) percentage expression of NF-jB, data are mean SEM of eight rats, *P < 0.05 versus control group, •P < 0.05 versus cadmium group without TQ treatment. ND, nondetectable; Cd, cadmium.
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higher serum testosterone level, and testicular GSH and SOD activity, and significantly lower levels of MDA, NO and cadmium ion in testicular homogenates as compared to the cadmium group nontreated with TQ (Table 1). Testicular histopathology Cadmium administration caused widespread necrosis and vacuolisation of the seminiferous tubular cells, associated with impaired spermatogenesis, interstitial tissue oedema and haemorrhages. However, TQ treatment markedly ameliorated the cadmium-induced damage of testicular tissue and preserved spermatogenesis in most of seminiferous tubules (Fig. 1). Also, Johnsen score was significantly decreased in cadmium group nontreated with TQ as compared to the control group. On the other hand, TQ treatment resulted in a significant increase in Johnsen score (Fig. 1). Testicular immunohistochemistry Significant increases in the expression of iNOS, TNF-a, COX-2, NF-jB and caspase-3 in the cells of seminiferous 6
tubules were observed in rats received cadmium chloride as compared to the control animals. On the other hand, TQ treatment significantly decreased the cadmiuminduced expression of iNOS, TNF-a, COX-2, NF-jB and caspase-3 in the cells of seminiferous tubules as compared to the cadmium group nontreated with TQ (Figs 2–6). The slides from the cadmium group without TQ treatment which were incubated with normal rabbit serum instead of the primary antibodies showed no staining at all indicating the specificity of the used antibodies (figures not shown). Discussion The present work, in agreement with previous studies, confirmed that oxidative stress, increased lipid peroxidation, depletion of antioxidant defences and increased production of pro-inflammatory mediators are implicated in the pathogenesis of cadmium-induced testicular toxicity (Aktas et al., 2012; Farombi et al., 2012; Fouad & Jresat, 2013). Also, the present study demonstrated that TQ treatment provided a significant protective effect against testicular injury caused by cadmium in rats as indicated by the improvement in the disturbed biochemical parameters and © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
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(d) Fig. 6 Immunohistochemical staining of caspase-3 in rat testes from: (a, 2009) control group showing no expression of caspase-3; (b, 2009 and inserted figure, 4009) cadmium group without thymoquinone (TQ) treatment showing a significant increase in caspase-3 immunoreactivity in the cytoplasm of seminiferous tubular cells; (c, 2009) TQ plus cadmium group demonstrating a significant reduction in caspase-3 immunostaining. Brown colour indicates caspase-3 positivity; (d) percentage expression of caspase-3, data are mean SEM of eight rats, *P < 0.05 versus control group, •P < 0.05 versus cadmium group without TQ treatment. ND, nondetectable; Cd, cadmium.
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amelioration of testicular tissue damage observed by histopathological and immunohistochemical examinations. Recent evidence suggests that cadmium-induced testicular toxicity occurs as a result of oxidative stress and increased generation of reactive oxygen species (Ji et al., 2012; Zhang et al., 2012). Increased production of reactive oxygen species depletes reduced glutathione and enhances lipid peroxidation in testicular tissue (Abarikwu et al., 2013; Spiazzi et al., 2013). Also, cadmium induces a cascade of inflammatory reactions with increased production of pro-inflammatory cytokines, particularly TNFa which is responsible for further testicular tissue injury (Al-Azemi et al., 2010; De Freitas et al., 2012). In addition, increased NO production in the testes is involved in the pathogenesis of cadmium toxicity (Gunnarsson et al., 2003; Fouad et al., 2009). It was reported that inflammatory stimuli, particularly TNF-a, can increase the expression of iNOS which catalyses the production of a large amount of NO. Excess NO reacts with superoxide anion to generate peroxynitrite radical responsible for cell damage by nitrating cellular macromolecules (Kobayashi, 2010). Also, excess NO depletes intracellular GSH and increases the susceptibility to oxidative stress (Ekici et al., © 2014 Blackwell Verlag GmbH Andrologia 2014, xx, 1–10
2012). Moreover, cadmium induces the expression of COX-2, which increases the production of inflammatory prostaglandins implicated in testicular tissue injury (Gunnarsson et al., 2003; Fouad et al., 2013). Thymoquinone is the main active constituent isolated from Nigella sativa seed oil and is known to have multiple biological effects. TQ exerts marked antioxidant activity, scavenges reactive oxygen radicals, suppresses lipid peroxidation and maintains the antioxidant defence mechanisms during free radical reactions (El-Khouly et al., 2012; Umar et al., 2012; Woo et al., 2012). Also, TQ inhibits iNOS activity and decreases NO production and therefore prevents nitrosative tissue stress (Ammar et al., 2011; Kanter, 2011). In addition, TQ exhibits significant anti-inflammatory activity because it reduces the release of pro-inflammatory cytokines (Vaillancourt et al., 2011; Umar et al., 2012) and inhibits COX-2 which is capable of producing large amounts of inflammatory prostaglandins (El Mezayen et al., 2006; Vaillancourt et al., 2011). This in accordance with the present results which revealed that TQ treatment significantly suppressed lipid peroxidation, prevented the depletion of GSH, decreased NO production and reduced the expression of iNOS, 7
Thymoquinone prevents cadmium testicular injury
TNF-a and COX-2 in the testes of rats exposed to cadmium toxicity. It was also reported that cadmium caused NF-jB activation with subsequent inflammatory reactions responsible for testicular tissue injury (Fouad & Jresat, 2013; Fouad et al., 2013). Elevated TNF-a is a well-known inducer of NF-jB signalling pathway (Li & Verma, 2002). The present results revealed that TQ significantly reduced the expression of testicular NF-jB in rats exposed to cadmium toxicity. This is in accordance with previous results which demonstrated that TQ provided a significant anti-inflammatory effect by inhibiting the activity of NF-jB (Chehl et al., 2009; Vaillancourt et al., 2011). Therefore, the testicular protective effect of TQ can be attributed to its ability to inhibit NF-jB signalling pathway which promotes the transcription of TNF-a, COX-2 and iNOS genes (Abd ElGhany et al., 2009; Chehl et al., 2009; Shen et al., 2013). The present study revealed that TQ significantly decreased the cadmium-induced expression of caspase-3, an executioner of cell apoptosis, in the testicular tissue. This is in agreement with previous studies which showed that TQ provided a significant anti-apoptotic effect by inhibiting caspase-3 activity (Helal, 2010; Ullah et al., 2012). Therefore, it could be stated that TQ protected against cadmiuminduced testicular cell apoptosis. The reduced caspase-3 activity observed with TQ treatment may be due to its free radical scavenging activity and anti-inflammatory action with reduced expression of TNF-a and NF-jB. Also, to the best of our knowledge, this is the first study to demonstrate the inhibitory effect of TQ on the expression of NF-jB, COX-2, TNF-a and caspase-3 in the testicular tissue. The present work revealed that TQ significantly attenuated the increase in testicular cadmium ion concentration resulted from cadmium chloride administration. This may be due to the ability of TQ to prevent cadmium-induced depletion of GSH which has antioxidant and metal-chelating activities (Rooney, 2007). The thiol groups in GSH can bind heavy metals with high affinity and play an important role in intracellular heavy metal detoxification (Satoh et al., 2000). It was concluded that TQ significantly abrogated cadmium-induced testicular injury and dysfunction in rats. The antioxidant and anti-inflammatory activities of TQ can be considered the main factors responsible for the testicular protective effect. Therefore, TQ may represent a potential therapeutic option to protect against the deleterious effect of cadmium exposure on the testes. References Abarikwu SO, Iserhienrhien BO, Badejo TA (2013) Rutin- and selenium-attenuated cadmium-induced testicular pathophysiology in rats. Hum Exp Toxicol 32:395–406.
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