2 , 3 , 7 , 8-tetrachloro-dibenzo-p-dioxin induced testicular toxicity in rats and the protective effect of quercetin : Biochemical , histopathological and immunohistochemical studies

Mohamed SA. El-Gerbed, Ahmed M. Abu El-Saad, Abdullah Bedeer Haussein Zoology Department, Faculty of Science, Damenhour University, Damenhour, Egypt. Department of Zoology, Faculty of Science, Alexandria University, Alexandria, Egypt. Department of Biology, College of Medicine, University of Dammam, Kingdom Saudi Arabia, 3Department of Zoology, Faculty of Science, Mansura University, Mansura, Egypt. Department of Biology, College of Medicine, University of Dammam, Kingdom Saudi Arabia.


INTRODUCTION
There has been increased awareness of the possible effects of environmental contaminants on male reproduction.An environmental agent should disrupt reproductive function in the male at several potential target sites, the most important being the testes, the male gonads, which are the sites of spermatogenesis and androgen production (Rebourcet et al., 2010).The risk to human health posed by exposure to these persistent organic pollutants has been one of worldwide concerns (Humblet et al., 2008;Collins et al., 2009).Numerous environmental toxicants have been shown to adversely affect spermatogenesis in rodents and humans which can lead to low sperm count, abnormal sperm morphology and poor semen quality (Sharpe et al., 2010;Wong et al., 2010).
2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin (TCDD) is the byproducts of various industrial processes (i.e., bleaching of paper the germ cells (Kim et al., 1999). . .Flavonoids are polyphenolic compounds with strong antioxidant properties widely distributed in dietary fruits; vegetables and herb medicine (Anjaneyulu et al., 2004).It is particularly abundant in onions (0.3 mg/g fresh weight) and tea (10-25 mg/L) (Scalbert et al., 2000).It has been reported that quercetin possess a number of biological effects such as its antioxidant properties against many diseases, including ischemic heart disease, atherosclerosis, liver fibrosis, renal injury and biliary obstruction (Ghosh et al., 2009;Liu et al., 2010).Also, quercetin possesses a lot of activities including anti-oxidative activity (Abdel-Raheem et al., 2009), anti-inflammation (Morales et al., 2006) and anti-apoptosis (Liu et al., 2010).Increasing evidence shows that quercetin can protect kidney from injury induced by nephrotoxins (Morales et al., 2006).And exhibits favorable hepatoprotective effect against ethanol hepatotoxicity by counteracting oxidative stress in vivo (Chen, 2010) and in vitro (Yao et al., 2008).Flavonoids can prevent oxidative damage as a result of their ability to scavenge reactive oxygen species such as hydroxyl radical and superoxide anion (Galati et al., 2002) and metal chelating (Pedrielli et al., 2002).The present investigation was carried out to explore the protective effect and the possible ameliorative role of quercetin on TCDD -induced reproductive toxicity.

Chemicals
TCDD was obtained from Accu Standard Inc. (New Haven, CT, USA).Quercetin and dimethyl sulfoxide (DMSO) was supplied by Merck (Germany).Olive oil and all other chemicals were supplied by Sigma Chemicals (St. Louis, MO, USA).

Animals
Adult sexually mature male albino rats (weighing 170 ± 6g; 90 days old) were purchased from the animal breeding colony of the High Institute of Public Health, Alexandria University, Alexandria, Egypt, were used.Rats were housed in clean polypropylene cages, and allowed to acclimatize to their new environment for 14 days prior to initiation of treatment.Throughout the study, this experiment was performed in a controlled environment (maintained at 23 ±3 °C, a relative humidity of 60 ±10 °C, in 12-h light-dark cycle).They were fed standard rat chow and tap water ad libitum.Experiments were initiated only when the feeding and drinking of the rats had stabilized at the normal level.

Experimental design
Rats were randomized into four experimental groups of 10 males.

GroupI:
The normal control group administration of olive oil once per day and administered via gavage for four weeks (28 days) of treatment.

Group II:
The TCDD treatment group received 27.5µg / kg TCDD (2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin once per day, which had been dissolved in acetone and then it was diluted with corn oil (1:6, v/v) in volumes of 5 ml/kg body weight.The acetone was evaporated under nitrogen before administration.TCDD was orally administered via gavage for four weeks.The test dose was determined on the basis of findings from previous studies (Pohjanvirta et al., 1993).
Group III: Co-treated TCDD with quercetin, rats were given quercetin dissolved in 0.5% DMSO (20 mg/kg bw per day) once per day via gavage (Kalender et al., 2012).Thirty minutes later, TCDD dissolved in olive oil (27.5 µg/ml) was administered, also via gavage for four weeks of treatment.
Group IV: administration for the quercetin treated group, the rats were given quercetin dissolved in 0.5% DMSO at a dose of 20 mg/kg bw once per day via gavage for four week of treatment.
All experiments in this study were performed in accordance with the guidelines for animal research from the National Institutes of Health and were approved by the Local Committee on Animal Research of the Alexandria University, Alexandria, Egypt.

Tissue preparation
At the end of the fourth week of treatment, testes of each rat were quickly excised, rinsed with a cold 1.15% potassium chloride, and weighed.One testis was homogenized in 50 mM Tris-HCl buffer (pH 7.4) containing 1.15% potassium chloride and the homogenate was centrifuged at 10,000 g for 15 min at 4 ºC and the supernatant immediately collected and processed for biochemical studies and enzyme activity assays.

Lipid peroxidation assay
Lipid peroxidation process is determined by the thiobarbituric acid (TBA) method which estimates the malondialdehyde formation (MDA) according to Nair and Turner (1984).Briefly, a 0.33 ml of testes homogenate was mixed well with 3 ml of TBA reagent.The mixture was incubated for 20 min in a boiling water bath.After cooling, the mixture was centrifuged at 3000g for 20 min.The supernatant was measured at 532 nm.Lipid peroxidation is expressed as n moles MDA/g tissue.

Reduced glutathione (GSH) assay
Levels of GSH were determined in the testes homogenates (10%) according to the Ellman (1959).One milliliter of supernatant was treated with 0.5 ml of Ellman's reagent (19.8mg of 5, 5`dithiobisnitro benzoic acid in 100 ml of 0.1% sodium citrate) and 3.0 ml of phosphate buffer (0.2 M, pH 8.0).The absorbance was read at 412 nm in spectrophotometer.To prevent the autoxidation of GSH, the samples were reduced with potassium borohydride prior to analysis (Kleinman and Richie, 2001).

Enzyme Assays
Superoxide dismutase (SOD) activity was determined by the method of Kakkar et al. (1984).Superoxide radicals react with nitroblue tetrazolium in the presence of NADH and produce formazan blue.SOD removes the superoxide radicals and inhibits the formation of formazan blue.The intensity of colour is inversely proportional to the activity of the enzyme.
The activity of catalase (CAT) was determined by the method of Sinha (1972).Dichromate in acetic acid was converted to perchromic acid and then to chromic acetate, when heated in the presence of H 2 O 2 .The chromic acetate formed was measured at 620 nm.Glutathione peroxidase (GSH-Px) was estimated by the method of Rotruck et al. (1973).A known amount of enzyme preparation was allowed to react with hydrogen peroxide in the presence of GSH for a specified time period.Then the remaining GSH was measured by the method of (Ellman, 1959).
The glutathione S-transferase (GST) activity was determined spectrophotometrically by the method of Habig et al. (1974) in which 1-chloro-2, 4-dinitrobenzenewas used as a substrate.Lactate dehydrogenase (LDH) activity was determined by the method of Wahlefeld (1983) using sodium-lactate and NAD as the substrate.The reaction was initiated by the addition of the substrate and the increase in absorbance at 340 nm resulting from the formation of NADH was used for the calculation for LDH activity.

Ascorbate determination
Ascorbate was determined according to Omaye et al. (1997) and expressed as mmol ascorbic acid/g wet tissue.

Protein assay
Contents of protein were measured in the testicular homogenates (10%) according to the method of Lowry et al. (1951) using bovine serum albumin as standard.

Evaluation of sperm characteristics
The spermatozoa were counted by the method described by Yokoi et al. (2003).Testicular sperm head numbers were assessed by the procedure of Meistrich and Van Beek (1993) to evaluate the numbers of mature elongate spermatids in the testis.The progressive spermatozoa motility was evaluated according to the method described by Zemjanis (1970).An aliquot of sperm suspension was stained using Wells and Awa's (0.2 g of eosin and 0.6 g of fast green dissolved in distilled water and ethanol in ratio 2:1) for sperm morphology and live-dead examination (Wells and Awa, 1970).Also, Two thousand sperms were evaluated on each slide and results were recorded as percentage of abnormal sperm for each slide.Abnormal heads and tails were evaluated using the criteria by Okamura et al. (2005) .

Hormonal assays
The blood samples were obtained from the animals by heart puncture.The serum level of testosterone was determined according to Abraham (1970); luteinizing hormone was detected according to Jaffe and Behrman (1974).

Histopathological examination
To assess histological changes, the fixed testicular tissues from 10 surviving rats in each group were paraffin embedded.Then, they were prepared as 4-mm-thick sections using a microtome.They were double stained with hematoxylin and eosin (Bancroft and Cook, 1994) and were observed by light microscope.

PCNA-Labeling Index (PCNA-LI)
Slides were examined under the light microscope with a magnification X 200 and thin sections were evaluated for PCNA immunostaining.Microscopic fields were chosen at random.Five fields per slide were evaluated.Only the basal germ cells of the seminiferous tubules were counted as the PCNA-LI for each seminiferous tubule and was estimated as a percentage of immunolabeled cells (cells with brown nuclear staining was positive PCNA-ir) to all basal cells according to (Tousson et al., 2011).

Statistical analysis
The data were expressed as the mean ± SE and were analyzed using SPSS 11.0 for windows.Statistical significance was calculated using one-way analysis of variance (ANOVA).Statistical evaluation of data was done following DMRT procedure for multiple comparisons.A difference was considered significant at p < 0.05.

Clinical findings
Death was not observed in any of the experimental groups during the treatment period.However, in dioxin treated group, few clinical signs such as alterations in feeding and drinking behaviors, wasting syndrome, reduced activity, increasing weakness, slight diarrhea and fur loss were observed.

Markers of oxidative damage
Testicular MDA concentration was significantly (p < 0.05) increased in the testis of rats treated with TCDD.In the quercetin + TCDD-treated animals, MDA level was significantly (p < 0.05) decreased compared with animals administered TCDD alone (Table 1).Our results indicated that the GSH content was decreased in TCDD or quercetin exposure alone (Table 1).
The decrease in the GSH content was over 30.4 % in the TCDD group and up to 27.2 % in the quercetin group relative to the corresponding control.Moreover, the TCDD-induced decrease of GSH in the testicular tissue was partially prevented by quercetin.The results shown in table 1 indicate that ascorbate content remained unaltered in the testicular tissue either when TCDD was administered alone or when co-administered with quercetin.The administration of quercetin alone had the tendency to increase ascorbate content in the testis.

Enzyme assay
The results obtained in this study show that TCDD exposure significantly (p < 0.05) decreased SOD activity but in the quercetin +TCDD animals, the SOD activity showed a significant reversal to the control value in the testicular tissue (Fig. 1).In animals that were administered quercetin alone, the testicular SOD activity was increased when compared with the corresponding control.Testicular CAT activity was significantly decreased (p < 0.05) in TCDD intoxicated rats.However, co-administration with quercetin showed a restorative response of CAT activity in the testes as values returned to that of control (Fig. 2).A significant increase (p < 0.05) in the activity of GSH-Px was observed in the testis in animals exposed to TCDD.In the quercetin +TCDD animals, this increase subsided, but was still higher than the corresponding control value in the testicular tissue.Quercetin alone increased the GSH-Px activity beyond the control value (p < 0.05) (Fig. 3).GST activity in the testicular tissue was significantly decreased in animals exposed to TCDD.Co-administration with quercetin partially restored GST activity.However, quercetin administration alone had no effect on GST activity (Fig. 4).Moreover, LDH activity increased significantly (p < 0.05) in the testicular tissue by TCDD exposure.Co-administration with quercetin normalized LDH to the control value.Interestingly, animals that received quercetin alone showed an increase in testicular LDH activity (Fig. 5).

Spermatological analysis
Administration of TCDD reduced the number of sperms in the testis.The motility of the spermatozoa dropped below the control value by 40 %.A significant increase in the number of abnormal and dead sperm was observed.These spermatological .variables in the quercetin + TCDD animals were not normalized to the corresponding control value.Although, there was a tendency for the recovery in the spermatozoa viability (vs TCDD.group, p < 0.05), quercetin administered alone had no effects on these parameters of spermatozoa function (Table 2).

Evaluation of Testosterone hormone and luteinizing hormone
Treating animals with TCDD for 4 weeks showed significant reduction (P < 0.05) in serum testosterone hormone and luteinizing hormone in comparison with control group (Fig. 6 a&b).Treatment with TCDD + Quercetin caused significant increase (P < 0.001) in serum testosterone and LH in comparison with rats treated with TCDD.There was insignificant increase in levels of these hormones in sera of animals treated with quercetin alone in comparison with control group.

Histopathological findings
Figures (7A-D) shows photomicrographs of testes from the different experimental groups.In control and quercetin groups, there was no evidence of testicular abnormality.Light microscopic examination revealed normal cytoarchitecture and maturation of germinal epithelium.The seminiferous tubules appeared uniform in size and shape.Leydig cells and blood vessels were found in the interstitial connective tissue between the tubules.A regular arrangement of spermatogenic epithelium existed in seminiferous tubules (Fig. 7A&D).
Testicular histopathology showed a detectable effect of TCDD on spermatogenesis (Fig. 7B) at the end of 4th week.Administration of dioxin caused severe histopathologic lesion.Seminiferous tubules showed loss of normal histoarchitecture widely separated from each other with loose arrangements due to reduction in diameter and atrophy of the tubules.The majority of the seminiferous tubules possessed irregular or ruptured basement membranes.Testicular atrophy leading to degenerative changes in the germinal epithelium, the testes with partial loss of the spermatogenic cells, Moreover, the germ cell arrangement within the seminiferous tubules was disrupted by exposure to dioxin.In the group III (quercetin plus TCDD) treated group, the majority of the seminiferous tubules possessed regular basement membranes with normal histoarchitecture, These tubules with no evidence of testicular abnormalities (Fig. 7 C).

Immunohistochemical results
Only the spermatogonia in control and quercetin groups (GI and GIV respectively) showed a positive strong reaction for PCNA-ir while the other spermatogenic cell types showed negative reaction (Fig. 8 A-D).The lumen of the seminiferous tubules in the control and quercetin groups was fully packed with sperms that showed a positive reaction for PCNA-ir (Fig. 8 A&D).Testes section in rats that treated with TCDD (G II) showed decreased in the numbers of spermatogonia that have PCNA-ir positive reaction as compared to control group (Fig. 8 B).Testes section in co-treated TCDD with quercetin showed increased in the numbers of spermatogonia that have PCNA-ir positive reaction as compared to TCDD group (Fig. 8 C).PCNA labeling index (PCNA-ir positive germ cells per tubule cross-section) were significantly decreased following TCDD treatment group (53±6%) in comparison to the control group (79±4%).On the other hand, PCNA labeling index were increased in testes section in co-treated TCDD with quercetin (63±5%) as compared to TCDD treatment group (53±6%) and decreased when compared with quercetin group (71±5 %).

DISCUSSION
Reproductive toxicology has been receiving increasing interest and concern in recent years (Amin, 2008).Environmental contaminants are known to induce reproductive toxicity by perturbing the prooxidant and antioxidant balance, which could induce oxidative stress (Elissa et al., 2011).TCDD is one of such environmental contaminants and has been shown to induce reproductive abnormalities in both wild life and humans by reduction in fertility (Sharpe et al., 2011).
Lipid peroxidation is a process of oxidative degradation of polyunsaturated fatty acids that result in impaired membrane structure and function (Goel et al., 2005).ROS, otherwise damage cellular lipids by binding to membrane anionic phospholipids (Sayed-Ahmed and Nagi, 2007), protein and DNA, finally the entire cell (Matés, 2000).Therefore, the increase in the testicular MDA in the present study could be due to the concomitant increase in the generation of ROS in TCDD treated group.Recent studies have shown that TCDD induced oxidative stress in mouse testis by decreasing enzymatic antioxidants, which resulted in enhanced level of ROS.This could impair testicular function in the mice (Jin et al., 2008).These data are consistent with our findings.TCDD treatment increased ROS generation and LPO and .decreased the specific activities of cellular antioxidant enzymes such as SOD, CAT, GST and the non enzymatic antioxidant GSH, indicating an imbalance in prooxidant and antioxidant system, leading to oxidative stress.ROS generated can cause DNA damage by directly attacking DNA or by activation of endonucleases that degrade DNA, which ultimately contribute to cell death (Floyd and Carney, 1992).Cells are equipped with antioxidant defense system to counteract the effect of ROS (Halliwell, 1994).Antioxidant enzymes are considered to be a primary defense that prevents biological macromolecules from oxidative damage.SOD is considered the first line of defense against deleterious effects of oxyradicals in the cell by catalyzing the conversion of superoxide anion to less dangerous H 2 O 2 , which is further degraded by CAT and GPx to water.CAT activity was significantly decreased.Decreased CAT activity may compromise the overall antioxidant enzyme defense system.CAT, along with GPx, exerts a major role in metabolizing hydrogen peroxide in cells and protecting cells against oxidative damage (Sun, 1990).Decrease in CAT levels would result in accumulation of hydrogen peroxide.Also, CAT and GPx protect SOD against effects of hydrogen peroxide (Marklund and Marklund, 1974).The decrease in activities of enzymatic antioxidants in TCDD-treated group may be related to increased LPO and ROS production.GSH is important in the regulation of cellular redox and a decline in its level is considered to be a good indicator of oxidative stress (Bray et al., 1993).This observation supports our finding where of a decline in GSH levels as evidenced by increased LPO.The decrease in testicular GSH level might be in part attributed to the inhibition of GR activity, which is responsible for regeneration of GSH from its oxidized form.GR is known to be inactivated by superoxide anion and hence, testicular cells showing reduced SOD activity might have enhanced flux of superoxide radicals that could potentially damage GR and decrease GSH content in these germ cells.All these antioxidant enzymes and GSH are extensively used as an index of unbalanced reactive oxygen species production and oxidative stress in physiological systems (Matés, 2000).Also TCDD decreased the antioxidant activity and increased the levels of hydrogen dioxide and MDA in the testis tissue in mice (Latchoumycandane et al., 2002).
In the present study, cell damage was evaluated by increase the activity of testicular lactate dehydrogenase in TCDD treated group.The hypersecretion of lactate may indicate a disturbance in the regulatory mechanism of intermediary metabolic pathway of Sertoli cells, finally resulting in detrimental effect on developing germ cells (Mita et al., 1982).However, an increase of lactate secretion by TCDD indicates a deleterious effect on the cell that may lead to lactate deficiency and infertility (Trejo et al., 1995).An expected effect of TCDD seems to be disturbing the physiological supply of lactate to the developing germ cells (Courtens and Plöen, 1999).The increase in LDH activity in the testicular tissue is usually viewed as an evidence of increased glycolysis and/or cell cytotoxicty (Raychoudhury and Kubinski, 2003).
Luteinizing hormone secreted by the anterior pituitary can affects Sertoli cells and Leydig cells and, in turn, increase the secretion of testosterone from Leydig cells.The secretion of LH is regulated by hormones secreted from the testes.This feedback loop maintains a balance in the level of androgenic hormone secretion (Yao, 2005).In the present study, it was observed that serum testosterone and LH had been significantly decreased by TCDD administered for four weeks in contrast to that of the vehicle control group.This finding is consistent with the results obtained from previous studies in male rats (Bookstaff et al., 1990).However, testosterone and LH of the blood plasma were not affected in rats exposed to TCDD (Li et al., 1997).This difference in the toxic effect was presumed to be due to the dissimilarity of the dose, duration of exposure and the species of animal.In the present study, suppression of serum testosterone is accompanied by a significant decline in the activities of antioxidant enzymes and increase in the levels of lipid peroxidation in the testis indicating excess ROS generation and induction of oxidative stress.The observed decline in the serum levels of testosterone following TCDD exposure may be due to adverse effect on the Leydig cells.In addition, the inhibitory effects of TCDD on pituitary luteinizing hormone (LH) secretion (Young et al., 2004) could also have contributed to the observed decline in serum testosterone levels.Several environmental toxicants have been reported to impair the testicular steroidogenesis via disruption of steroidogenic acute regulatory (StAR) protein expression and or by altering the activities of steroidogenic enzymes like cytochrome P450scc, 3βhydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase (Saradha et al., 2008).Oxidative changes in primary cultured Leydig cells have been observed following exposure to 2-bromopropane, an intermediate used in the production of pesticides, and cadmium which resulted in decreased testosterone secretion (Yang et al., 2003).Exposure of adult male rats to a single intraperitoneal injection of TCDD resulted in a dose-dependent reduction in Leydig cell volume per testis, which indicates Leydig cell as a potential target for TCDD toxicity (Johnson et al., 1992).TCDD has been shown to inhibit human chorionic gonadotropin (hCG)-induced testosterone secretion in rat Leydig cell primary cultures by decreasing the expression of steroiodogenic enzymes (StAR, P450scc and 3β-hydroxysteroid dehydrogenase) and cellular levels of cAMP (Lai et al., 2005).TCDD has antiandrogenic effects on both humans and animals, and it could therefore affect the form and function of testes.TCDD inhibited the growth and differentiation of seminal vesicle epithelium during postnatal development (Hamm et al., 2005) and caused atrophy and decreases in the diameter of the seminiferous tubules and in the spermatogonial population (El-Sabeawy et al., 1998).It also caused the feminization of male mice and as well as their sexual dysfunction (Gray et al., 1997).An epidemiology study found that the testosterone level had a negative correlation with serum TCDD, and the number of spermatozoa decreased to half that of the normal levels in men exposed chronically to TCDD (Egeland et al., 1994).In the present study, we found that TCDD resulted in a decrease in the spermatozoa number and sperm motility and increase in dead and abnormal sperm rate indicative damage of the testis structure in rats.Most toxic effects induced by TCDD were mediated by binding to the AhR, which binds together with a second protein, aryl hydrocarbon receptor nuclear translocator, to the response elements of a number of target genes and thus modulates gene expression (Nau, 2006).The activated AhR induces the expression of various genes with xenobiotic response elements in their enhancer regions, such as the gene for cytochrome P450.Esser et al. (2005) found that TCDD interfered with the physiological signaling of the AhR, led to cell-specific changes in gene transcription and cell differentiation, and thereby producing toxic effects.TCDD combined with AhR, and thereby induced a change in the expression of monoamine oxidase related to CYP450 enzymes activated by AhR (Poland and Kimbrough, 1984).TCDD could have caused the decrease in testosterone by damaging the smooth endoplasmic reticulum or mitochondria of the Leydig cells, thereby interfering with enzymes involved in androgen synthesis (Roman et al., 1995).Kim et al. (1999) reported that TCDD may cause testicular morphological lesions and a decrease in steroidogenesis.Similarly, in this study, treatment of TCDD caused morphological and immunohistochemical changes.Seminiferous tubules, where only the Sertoli cells exist with few germ cells found, were shown to be necrosed and completely damaged.Thus, it suggests that the toxicity of TCDD in the testicles caused abnormal spermatogenesis.There is much concern that exposure to environmental contaminants can produce major pathological effects in the reproductive systems of both humans and animals.2, 3, 7, 8-TCDD is the most toxic environmental contaminant known and has been shown to induce reproductive abnormalities (Chia, 2000).
The current study showed histopathologic changes in the seminiferous tubules of testis ranging from vascular congestion to focally diffuse interstitial edema, focal areas of tubular degeneration and coagulative necrosis of spermatozoa were observed following dioxin administration to the experimental rats.Furthermore, interstitial tissue in the treated rats showed a relative increase in edema of the interstitium and congested blood vessels suggesting inflammation.In addition, there were apoptotic signs in Leydig cells.Later, seminiferous tubules were relatively widely separated from each other with fragments of degenerated peritubular tissue detected inbetween.In agreement with the present results, TCDD was found to produce atrophy, morphological changes, impaired spermatogenesis, and epididymal lesions in the testes of experimental animals (Al-Bayati et al., 1988).Further, when 2,3,7,8-TCDD was administered to rats, histopathologic examination revealed a decrease in the diameter of seminiferous tubules and the number of testicular sperm (El-Sabeawy et al., 1998).
Antioxidants are exogenous or endogenous compounds acting in several ways, including scavenging reactive oxygen species or their precursors, inhibiting ROS formation, and binding metal ions needed for the catalysis of ROS generation (Bhatt and Flora, 2009).Flavonoids can prevent oxidative damage as a result of their ability to scavenge reactive oxygen species such as hydroxyl radical and superoxide anion (Galati et al., 2002).The antioxidant properties of flavonoids depend on both metalchelating properties and free radical scavenging of reactive oxygen species (Pedrielli and Skibsted, 2002).Quercetin is one of the most frequently studied dietary flavonoids, distributed in vegetables, fruits and many other dietary sources (Bhatt and Flora, 2009).Quercetin, a potent anti-oxidant, scavengers' free radicals directly, inhibits lipid peroxidation and alters anti-oxidant defence pathway in vivo and in vitro (Anjaneyulu and Chopra, 2004).Previous studies have shown that flavonoids have protective effects against oxidative damage induced by organophosphorous pesticides (Panemangalore and Bebe, 2009;Sadowska, 2010).In addition, it has been reported that quercetin has protective effects on antioxidant enzyme metabolism and histopathological changes induced by chlorpyrifos in rat lung tissues (Uzun et al., 2010).Our results clearly showed that quercetin treatment successfully prevent oxidative and histological damage caused by TCDD.
When TCDD and quercetin given together, quercetin reduced lipid peroxidation induced by TCDD treatment via decreasing the levels of MDA in testicular tissue of TCDD-treated rats.Although there are a few studies about the protective effect of quercetin against testicular toxicity by TCDD, there are many studies about the antioxidant properties of quercetin against lipid peroxidation (Pushpavalli et al., 2010). Recently Ciftci et al. (2012) demonstrated that quercetin induced the testicular antioxidant enzyme capacity in TCDD intoxicated rats.However, our results in the present work included a mutibiochemical markers concerning testicular damage such as assays of ascorbate content, GST, LDH activities, luteinizing hormone level, in addition to immunohistochemical studies which were not included in the previous work of Ciftci et al. (2012).Our results confirmed the findings of this earlier study concerning the toxic effects of TCDD in testicular tissue and protective role of quercetin.Renugadevi and Prabu (2009) determined that quercetin treatment significantly protected kidney tissue of rat against toxicity of cadmium by decreasing TBARS formation.These results are in agreement with our findings and confirmed our study.Quercetin treatment in the current study prevented a decrease in GSH level and the activities of SOD, CAT and GST in testis by TCDD.Similarly, Abdel-Raheem et al. (2009) showed as many other studies that quercetin treatment induced detoxifying enzymes and thereby prevented toxicity in different organs of rats induced by gentamicin.On the other hand, it was indicated that quercetin treatment together with TCDD prevented adverse effects of TCDD in terms of histological alternations.Similarly, Yousef et al. (2010) showed that quercetin treatment exerts potent protective effects in histological damage caused by different agents such as cadmium, paracetamol and ethanol.These beneficial effects (in terms of oxidative and immunohistological results) of quercetin on testicular toxicity of TCDD are related with their high antioxidant capacity.
In conclusion, the current study demonstrated that exposure to TCDD caused deterioration in semen quality, decrease of fertility indexes as well as alternation in lipid profile and decreased the enzyme activities of serum.Moreover, the testosterone decreased and there were many histological changes in the tests.Our results reveal that quercetin has a marked ameliorative effect on testis toxicity, as rats receiving this flavonoid in addition to TCDD.Therefore, the present study elucidated the therapeutic effects of quercetin administered in combination with TCDD to minimize its reproductive toxicity.

Fig. 1 :
Fig. 1: Effects of quercetin on TCDD-induced changes in superoxide dismutase (SOD) activity in the testis of rats.Values are mean ± SE; n=7.Values not sharing common superscript letters (a-c) differ significantly at p<0.05 (DMRT).

Fig. 2 :
Fig. 2: Effects of quercetin on TCDD-induced changes in catalase (CAT) activity in the testis of rats.Values are mean ± SE; n=7.Values not sharing common superscript letters (a-c) differ significantly at p<0.05 (DMRT).

Fig. 3 :
Fig. 3: Effects of quercetin on TCDD-induced changes in Glutathione peroxidase (GSH-Px) activity in the testis of rats.Values are mean ± SE; n=7.Values not sharing common superscript letters (a-c) differ significantly at p<0.05 (DMRT).

Fig. 4 :
Fig. 4: Effects of quercetin on TCDD-induced changes in glutathione Stransferase (GST) activity in the testis of rats.Values are mean ± SE; n=7.Values not sharing common superscript letters (a-c) differ significantly at p<0.05 (DMRT).

Fig. 5 :
Fig. 5: Effects of quercetin on TCDD-induced changes in Lactate dehydrogenase (LDH) activity in the testis of rats.Values are mean ± SE; n=7.Values not sharing common superscript letters (a-c) differ significantly at p<0.05 (DMRT).

Fig. 6 :
Fig. 6: Effects of quercetin on TCDD-induced changes in serum level of (a) testosterone hormone and (b) luteinizing hormone in rats.Values are mean ± SE; n=7.Values not sharing common superscript letters (a-c) differ significantly at p<0.05 (DMRT).

Fig. 7 :
Fig. 7: Photomicrographs of testicular sections from control and other experimental groups.In control (Fig. A) and, quercetin (Fig. D) -treated groups showing The seminiferous tubules were well developed with active spermatogenesis without any structural changes.Section of testes from rat treated with TCDD (Fig.B).Seminiferous tubules possess irregular basal lamina and are separated from each other.Also, wide gaps between neighboring cells and enlargement of the intercellular spaces.Interstitial tissues showed edema, hemorrhage and vacuolation.Many seminiferous tubules were edematous with intact germinal layer and undergoing degeneration along with loss of spermatogenesis.Cellular alterations at different spermatogenic stages.In quercetin plus TCDD (Fig.C) treated groups, the normal tubules were seen, Abbrev: seminiferous tubules (St), elongated spermatids (Sp), Leydig cells (Ly), lumen (L), vacuole (V)

Fig. 8 :
Fig. 8: Photomicrographs of PCNA-ir (immunoperoxidase labelling) in the testes cross sections from control and other experimental groups.Control (Fig. A) and quercetin (Fig. D) -treated groups showing different stages of spermatogenic cells in the seminiferous tubules with normal distribution of spermatogonia in seminiferous tubules of rats were seen.(Fig. B) Section of testes from rat treated with TCDD, showing only a few numbers of spermatogonia have negative reaction for PCNA-ir.Note that the lumen of the tubules was clear and the spermatozoa were not seen.(Fig. C) showings, few numbers of the spermatogonia in the seminiferous tubules have positive reaction for PCNA.Note, normal distributions of spermatogonia in seminiferous tubules of quercetin plus TCDD rats were seen.

Table 1 :
Effect of quercetin on the lipid peroxidation levels and antioxidant defence system in testicular tissue of rats treated with TCDD .Values are mean ± SE for ten rats in each group.Values within rows with no common superscripts are significantly different (LSD multiple range test, p<0.05).

Table 2 :
Effect of TCDD and quercetin on spermatozoa parameters .Values are mean ± SE for ten rats in each group.Values within rows with no common superscripts are significantly different (LSD multiple range test, p<0.05).