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Abstract

The aim of the present study was to investigate the potential oxidative damage of di-(2-ethylhexyl) phthalate (DEHP) in the rat testis and to further elucidate the potential modulatory effect of quercetin. DEHP was diluted in corn oil and given to rats by oral gavage at doses 0, 300, 600, and 900 mg/kg/day (groups I, III, IV, or V, respectively) for 15 consecutive days. Group VI was pretreated with quercetin (90 mg/kg), 24 h before starting the experiment and then treated with DEHP (900 mg/kg/day) for 15 consecutive days. Group II was treated with quercetin (90 mg/kg/day). The relative testes weight and sperm motility were significantly decreased by treatment with 900 mg/kg of DEHP. Both sperm count and daily sperm production were significantly decreased by DEHP treatment at doses of 600 and 900 mg/kg. Serum testosterone level and prostatic acid phosphatase (ACP) activity and testicular lactate dehydrogenase-X (LDH-X) activity were significantly decreased in animals treated with 900 mg/kg. Serum total ACP activity was significantly increased in animals treated with 600 and 900 mg/kg of DEHP. DEHP treatment induced oxidative stress and histopathological abnormality. These abnormalities were effectively normalized by pretreatment with quercetin except for LDH-X near normalcy. In conclusion, the findings of this study demonstrate that DEHP impairs testicular function at least, in part, by inducing oxidative stress and quercetin has a potent protective effect against DEHP-induced testicular toxicity in rats.

Keywords

DEHP quercetin testis spermatogenesis oxidative stress

Introduction

Phthalic esters are compounds widely used as plasticizers. About 3 million tons of phthalic esters per year are produced worldwide and they can be found in many everyday products, such as PVCs, plastic bags, food packaging, cosmetics, industrial paints as well as blood transfusion packs.¹ Due to the non-covalent nature of their link with plastics, phthalates can easily leach from these products and, consequently, be ingested.² Very large proportion of the literature on the endocrine disruptors categorized as anti-androgens deals with phthalates.³ Di-(2-ethylhexyl) phthalate (DEHP) is currently the most commonly used phthalate plasticizer.⁴ After exposure, DEHP is rapidly hydrolyzed by esterases in the gut, liver, and blood into mono-(2-ethylhexyl) phthalate (MEHP), which is believed to be the active molecule.⁵

Numerous studies have been performed to assess the effects of phthalates, principally on the male reproductive functions. Exposure to phthalate esters results in inhibition of testicular testosterone synthesis associated with the development of abnormalities of the reproductive tract of fetal and prepubertal rat. In postpubertal rats, phthalates were also found to decrease sperm production.^6
–8 DEHP and its main metabolite, MEHP have been deleterious on the male reproductive system of the neonatal, prepubertal, and adult animals and induced dramatic changes in germ cells.^9,10 Although the induction of oxidative stress may represent a common mechanism in endocrine disruptor-mediated dysfunction, especially on testicular cells,¹¹ recent studies are also providing supporting evidences for such an effect with DEHP .^12
–14

Therapeutic strategies aimed at preventing or delaying generation of reactive oxygen species (ROS) might be a reasonable choice against DEHP-induced toxicity.¹⁵ Dietary intake of antioxidants is a plausible and effective way to augment and fortify endogenous defense systems, since many antioxidants can act as free radical scavengers resulting in cytoprotection.¹⁶ Quercetin, one of the most widely distributed flavonoids in plants including various vegetables, fruits, and herb medicine, is generally regarded as a prominent ROS scavenger.¹⁷ Quercetin offers protection against oxidative stress and cell death induced by a variety of insults in many types of cells.¹⁸ Quercetin has been reported to protect testicular cells from oxidative damage induced by environmental chemicals.¹⁹

Concern has increased regarding the adverse effects of DEHP on reproduction.²⁰ However, the mechanism by which phthalates and particularly DEHP exerts toxic effects in reproductive system are not yet fully elucidated. Therefore, the aim of the present study was to investigate the potential oxidative damage of DEHP in the rat testis and to further elucidate the potential modulatory effect of quercetin against this environmental pollutant.

Materials and methods

Chemicals

DEHP, quercetin, and DL-α hydroxycaproic acid were purchased from Sigma-Aldrich Chemical Company (St Louis, MO, USA). All other chemicals are of analytical grade.

Animals and treatment

Sixty adult male Wistar rats (220 ± 10 g) were housed in clean polypropylene cages and maintained on a 12-h light/12-h dark cycle and a temperature of 20–25°C with ad libitum access to food and water.

The animals were divided into six groups. Group I served as control receiving corn oil through the experiment. Group II received quercetin (90 mg/kg) suspended in corn oil and administered by oral gavage through the experiment. DEHP was diluted in corn oil and given to rats by oral gavage at doses 300, 600, or 900 mg/kg/day for 15 consecutive days (groups III, IV, and V, respectively). Group VI was pretreated with quercetin (90 mg/kg), 24 h before starting the experiment and then treated with DEHP (900 mg/kg/day) for 15 consecutive days. The doses and duration of treatment of DEHP and quercetin were selected as per previous publications.^21
–23 This study involving experimental animals was conducted in accordance with national and institutional guidelines for the protection of animal welfare.

Necropsy

Twenty-four hours after the last dose, the animals’ body weights were recorded and blood samples were collected from the retro-orbital sinus, under ether anesthesia, and allowed to clot at room temperature. Samples were centrifuged and supernatant serum was separated from the clot as soon as possible and stored at −80°C until total acid phosphatase (ACP) (TACP) and prostatic ACP (PACP) and testosterone assay. The animals were euthanized under anesthetic ether. The testes were removed, cleaned from adhering fat and connective tissues and weighed in grams. The relative testes weight (weight of testes (g)/body weight (g) × 100) was calculated. The cauda epididymides were used for sperm count and motility. One testis from each rat (the right) was processed for evaluation of daily sperm production (DSP), whereas the left one was homogenized in ice-cold 0.15 M KC1 (10% w/v). The homogenate was centrifuged at 9000g for 10 min at 4°C and the supernatant immediately collected and processed for biochemical studies and enzyme activity assays. One testis from each group was processed for histopathological examination.²⁴ Protein concentrations were determined using a bicinchoninic acid kit (Pierce, Rockford, Illinois, USA) that employed bovine serum albumin as a standard.

Sperm count and motility

Epididymides were dissected out, immediately minced in 5 ml of physiological saline and then incubated at 37°C for 30 min to allow spermatozoa to leave the epididymal tubules. The percentage of motile sperm was recorded using a phase contrast microscope at a magnification of 400×. Total sperm number was determined using a Neubauer hemocytometer as previously described.²⁵ To determine sperm motility, 100 sperms each were observed in three different fields, and classified into motile and nonmotile sperms, and the motility was expressed as percentage incidence.

Daily sperm production

DSP was determined as previously described.²⁶ The right testis was decapsulated and homogenized in 50 ml of ice-cold 0.9% sodium chloride solution containing 0.01% Triton X-100 using a Polytron homogenizer (Kinematica, Inc., USA).²⁷ The homogenate was allowed to settle for 1 min and then was gently mixed, and a 10 ml aliquot was transferred to a glass vial and stored on ice. After thorough mixing of each sample, the number of sperm heads (step 19 spermatid head) in four chambers of Neubauer type hemocytometer was counted under a light microscope with 40× objective. To calculate DSP, the number of spermatids at stage 19 was divided by 6.1, which is the number of days of the seminiferous cycle in which these spermatids are present in the seminiferous epithelium.

Serum testosterone

Testosterone was measured using the Pathozyme Testosterone ELISA kit (DRG international, Inc., USA). Briefly, standards, specimens, and controls were dispensed into appropriate wells, followed by testosterone horseradish peroxidase reagent and anti-testosterone reagent, before mixing thoroughly and incubating at 37°C for 90 min. Wells were then rinsed with deionized water and substrate solution was dispensed into each well, gently mixed, and incubated for 20 min. The reaction was stopped with the “stop reagent” and the absorbance recorded at 450 nm.²⁸

Serum TACP and PACP activity

α-Naphthylphosphate is hydrolyzed by ACP to phosphate and α-naphthol, which is converted with fast red TR salt into azodye. The increase in the absorbance of the azodye at 405 nm is proportional to TACP activity in the sample. PACP reaction can be blocked by tartarate and determined indirectly (through the non-prostatic ACP) by calculating the difference in the activities.²⁹

Testicular LDH-X

Testicular lactate dehydrogenase-X (LDH-X) activity was measured using α-ketovaleric acid as the substrate.³⁰ The activity of LDH-X is expressed as micromoles of NADH oxidized per minute per milligram protein.

Testicular oxidative stress status

The cellular lipid peroxidation (LPO) products within the testis were determined using thiobarbituric acid reactive substances during an acid-heating reaction. The samples were diluted by 1.5 ml trichloroacetic acid (20% w/v) and centrifuged at 3000g for 10 min. Then, the precipitation was dissolved in sulfuric acid and 1.5 ml of the mixture was added to 1.5 ml of thiobarbituric acid (TBA; 0.2% w/v). The mixture was then incubated for 1 h in a boiling water bath. Following incubation, 2 ml of n-butanol was added. The solution was centrifuged, cooled, and the absorption of the supernatant was recorded at 532 nm. The calibration curve of tetraethoxypropane standard solutions was used to determine the concentrations of TBA–malondialdehyde adducts in samples.³¹

Superoxide dismutase (SOD) was assayed by the method of Marklund and Marklund.³² Briefly, the assay mixture contained 2.4 ml of 50 mM tris(hydroxymethyl)aminomethane–hydrochloric acid buffer containing 1 mM ethylenediaminetetraacetic acid (pH 7.6), 300 µl of 0.2 mM pyrogallol, and 300 µl testis homogenate. The decrease in absorbance was measured immediately at 420 nm against blank at 10 s intervals for 3 min on a spectrophotometer. Catalase (CAT) was assayed as previously mentioned.³³ Briefly, the assay mixture contained 2.40 ml of phosphate buffer (50 mM, pH 7.0), 10 µl of 19 mM hydrogen peroxide (H₂O₂), and 50 µl testis homogenate. The decrease in absorbance was measured immediately at 240 nm against blank at 10 s intervals for 3 min on a spectrophotometer. The level of reduced glutathione (GSH) was determined as previously described based on the reaction with Ellman’s reagent (19.8 mg dinitrothiocyanobenzene in 100 ml 0.1% sodium citrate).³⁴ The absorbance was recorded at 412 nm using a spectrophotometer. The GSH content was expressed as micrograms per milligram protein.

Statistical analysis

Differences between obtained values (mean ± SD, n = 10) were compared by one-way analysis of variance followed by the Tukey–Kramer multiple comparison test. Comparison is made between (i) control (i.e. group I) and other groups and (ii) group V and group VI. Differences were considered statistical significance from at *p < 0.05; **p < 0.01; ***p < 0.001.

Results

Relative testes weight and sperm parameters

Table 1 shows the changes in relative testes weight and sperm parameters. Treatment of male rats (group V) with DEHP (900 mg/kg) significantly decreased the relative testes weight (p < 0.05), while DEHP at doses 300 and 600 mg/kg did not show any significant change as compared to the corresponding control. Pretreatment with quercetin (group VI) reverted the relative testes weight to normalcy (p < 0.05).

Table 1.

Effect of DEHP and/or quercetin on relative testes weight and sperm parameters.

Parameter	Group I	Group II	Group III	Group IV	Group V	Group VI
Relative testes weight [weight of testes (g)/body weight (g)x 100]	1.2 ± 0.068	1.26 ± 0.061	1.17 ± 0.042	1.17 ± 0.039	0.97 ± 0.063^a*	1.21 ± 0.037^b*
Cauda sperm count (x10⁶/rat)	80.32 ± 1.78	83.7 ± 2.14	77.53 ± 2.47	61.71 ± 4.87^a**	52.78 ± 4.89^a***	80.07 ± 4.3^b***
Sperm motility (x10⁶/rat)	60.89 ± 1.69	64.61 ± 1.95	61.05 ± 1.84	54.33 ± 4.55	41.56 ± 3.98^a***	55.51 ± 1.94^b*
Daily sperm production (x10⁶/testis/day)	36.14 ± 1.65	35.55 ± 2.14	34.1 ± 1.29	26.59 ± 1.5^a**	20.67 ± 1.09^a***	34.33 ± 1.4^b***

Group I: Control (vehicle), Group II: quercetin (90 mg/kg), Group III: DEHP (300 mg/kg/day), Group IV: DEHP (600 mg/kg/day), Group V: DEHP(900 mg/kg/day), Group VI: quercetin (90 mg/kg/day) + DEHP (900 mg/kg/day). DEHP: Diethylhexyl phthalate. Data are expressed as mean ± SD (n = 10). Comparison is made between: ^aGroup I and Groups II, III, IV, V, VI; ^bGroup V and Group VI by one way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparison test. The symbol represents statistical significance from control: *p < 0.05; **p < 0.01; ***p < 0.001.

Sperm count was significantly decreased by 23.17 and 34.29% in response to treatment with DEHP (600 and 900 mg/kg) (groups IV and V, respectively), while DEHP at a dose of 300 mg/kg (group III) did not show any significant change in sperm count as compared to the corresponding control. DEHP at a dose of 900 mg/kg (group V) decreased sperm motility by 31.75%, while the other doses did not show any significant change (groups III and IV) as compared to the corresponding control. Moreover, DSP showed significant reduction by 26.43 and 42.81% in response to treatment with DEHP (600 and 900 mg/kg) (groups IV and V, respectively), while it did not show any significant change at a dose of 300 mg/kg (group III) of DEHP as compared to the corresponding control. Pretreatment with quercetin (group VI) reverted the values of sperm count, motility, and DSP to normalcy (p < 0.001, p < 0.05, and p < 0.001, respectively, as compared to group V).

Serum testosterone and ACP (total and prostatic)

Animals treated with DEHP (900 mg/kg; group V) showed significant decrease by 69.46% in serum testosterone content as compared to the related control (Figure 1). DEHP at doses 300 and 600 mg/kg did not show any significant change in testosterone level (groups III and IV). Quercetin treatment (group II) significantly increased testosterone content (p < 0.01) as compared to the corresponding control. Animals pretreated with quercetin (group VI) showed normal testosterone content (p < 0.001, as compared to group V).

Figure 1.

Effect of DEHP and/or quercetin on serum testosterone. Group I: control (vehicle); group II: quercetin (90 mg/kg); group III: DEHP (300 mg/kg/day); group IV: DEHP (600 mg/kg/day); group V: DEHP (900 mg/kg/day); and group VI: quercetin (90 mg/kg/day) + DEHP (900 mg/kg/day). Data are expressed as mean ± SD (n = 10). Comparison is made between: (a) group I and groups II, III, IV, V, and VI; (b) group V and group VI by one-way analysis of variance followed by the Tukey–Kramer multiple comparison test. **p < 0.01: statistical significance from control; ***p < 0.001: statistical significance from control. DEHP: di-(2-ethylhexyl) phthalate.

TACP activity showed significant increase by 25.23 and 50.45% in response to DEHP (600 and 900 mg/kg, respectively) treatment as compared to the corresponding control (Figure 2). DEHP at a dose of 300 mg/kg did not show any significant change. Pretreatment with quercetin (group VI) normalized TACP content (p < 0.001, as compared to group V). Animals treated with 900 mg/kg of DEHP (group V) showed significant reduction by 42.86% in PACP content as compared to the corresponding control (Figure 3). DEHP at doses of 300 and 600 mg/kg did not show any significant change in PACP activity (groups III and IV). Pretreatment with quercetin (group VI) reverted PACP content toward normalcy (p < 0.05, as compared to group V).

Figure 2.

Effect of DEHP and/or quercetin on serum TACP. Group I: control (vehicle); group II: quercetin (90 mg/kg); group III: DEHP (300 mg/kg/day); group IV: DEHP (600 mg/kg/day); group V: DEHP (900 mg/kg/day); and group VI: quercetin (90 mg/kg/day) + DEHP (900 mg/kg/day). Data are expressed as mean ± SD (n = 10). Comparison is made between: (a) group I and groups II, III, IV, V, and VI; (b) group V and group VI by one-way analysis of variance followed by the Tukey–Kramer multiple comparison test. *p < 0.05: statistical significance from control; ***p < 0.001: statistical significance from control. TACP: total acid phosphatase; DEHP: di-(2-ethylhexyl) phthalate.

Figure 3.

Effect of DEHP and/or quercetin on serum PACP. Group I: control (vehicle); group II: quercetin (90 mg/kg); group III: DEHP (300 mg/kg/day); group IV: DEHP (600 mg/kg/day); group V: DEHP (900 mg/kg/day); and group VI: quercetin (90 mg/kg/day) + DEHP (900 mg/kg/day). Data are expressed as mean ± SD (n = 10). Comparison is made between: (a) group I and groups II, III, IV, V, and VI; (b) group V and group VI by one-way analysis of variance followed by the Tukey–Kramer multiple comparison test. *p < 0.05: statistical significance from control; **p < 0.01: statistical significance from control. PACP: prostatic acid phosphatase; DEHP: di-(2-ethylhexyl) phthalate.

Testicular LDH-X

Treatment with DEHP at a dose of 900 mg/kg significantly decreased LDH-X activity by 44.87% (group V), while at doses of 300 and 600 mg/kg, DEHP did not show any significant change (groups III and IV) as compared to the corresponding control (Figure 4). Pretreatment with quercetin (group VI) ameliorated LDH-X activity toward normalcy but did not reach normal level (p < 0.05, as compared to the corresponding control).

Figure 4.

Effect of DEHP and/or quercetin on testicular LDH-X activity. Group I: control (vehicle); group II: quercetin (90 mg/kg); group III: DEHP (300 mg/kg/day); group IV: DEHP (600 mg/kg/day); group V: DEHP (900 mg/kg/day); and group VI: quercetin (90 mg/kg/day) + DEHP (900 mg/kg/day). Data are expressed as mean ± SD (n = 10). Comparison is made between: (a) group I and groups II, III, IV, V, and VI; (b) group V and group VI by one-way analysis of variance followed by the Tukey–Kramer multiple comparison test. *p < 0.05: statistical significance from control; ***p < 0.001: statistical significance from control. DEHP: di-(2-ethylhexyl) phthalate.

Oxidative stress status

As shown in Table 2, animals treated with 900 mg/kg of DEHP (group V) showed increase in LPO by 66.4%, and reduction in the activities of SOD and CAT by 39.24 and 69.84%, respectively (group V), as compared to the corresponding control. DEHP at doses 300 and 600 mg/kg (groups III and IV) did not show any significant change in LPO or in the activity of either SOD or CAT. Pretreatment with quercetin (group VI) significantly suppressed (p < 0.001, as compared to group V) LPO. Pretreatment with quercetin (group VI) also reverted the reduction in SOD and CAT activities induced by treatment with DEHP (900 mg/kg/day) to normal value. GSH level was significantly decreased by 33.54 and 40.37% in response to DEHP treatment (600 and 900 mg/kg; groups IV and V, respectively) as compared to the related control. Pretreatment with quercetin (group VI) normalized (p < 0.01, as compared to group V) GSH level. Notable, animals treated with quercetin (group II) showed significant increase (31.06%) in GSH level as compared to the related control.

Table 2.

Effect of DEHP and/or quercetin on oxidative stress status.

Parameter	Group I	Group II	Group III	Group IV	Group V	Group VI
LPO (nmol of MDA/g testis)	45.71 ± 4.29	43.05 ± 4.11	46.55 ± 3.24	49.79 ± 3.18	76.06 ± 3.21^a***	48.88 ± 3.37^b***
SOD (U/g testis)	1.58 ± 0.04	1.79 ± 0.05	1.52 ± 0.07	1.41 ± 0.08	0.96 ± 0.06^a***	1.52 ± 0.05^b***
CAT (U/g testis)	3.68 ± 0.26	4.61 ± 0.29	3.79 ± 0.3	2.97 ± 0.27	1.11 ± 0.09^a***	3.43 ± .3^b***
GSH (µmol/g testis)	1.61 ± 0.08	2.11 ± 0.2^a*	1.44 ± 0.06	1.07± 0.04^a*	0.96 ± 0.03^a**	1.6 ± 0.14^b**

Histopathological examination

Specimens from control animals showed normal seminiferous tubules and spermatogenesis (Figure 5(a)). Similarly, quercetin showed virtually almost the same histologic architecture as control animal (Figure 5(b)). DEHP, however, induced degeneration of spermatogonial cells in some seminiferous tubules (300 mg/kg; Figure 5(c), arrow), degeneration and necrosis with pyknosis in the nuclei of spermatogonial cells and spermatids (600 mg/kg; Figure 5(d)), and azospermia in some seminiferous tubules (900 mg/kg) (Figure 5(e)). Prior administration of quercetin a head of DEHP challenge apparently ameliorated the histologic alterations in the seminiferous tubules compared to DEHP-treated rats (Figure 5(f)).

Figure 5.

Representative illustrations of histological morphology of rat testes. (a) Testicular cross section from control rats showing normal seminiferous tubules and spermatogenesis (H and E, ×40). (b) Testicular cross sections from quercetin-treated rats showing normal histologic architecture of seminiferous tubules and spermatogenic stages (arrow) (H and E, ×40). (c) Testicular cross sections from DEHP (300 mg/kg/day)-challenged rats showing degeneration of spermatogonial cells (arrow) in some seminiferous tubules (H and E, ×40). (d) Testicular cross sections from DEHP (600 mg/kg/day)-treated rats showing degeneration and necrosis with pyknosis in the nuclei of spermatogonial cells and spermatids (arrow) (H and E, ×160). (e) Testicular cross sections from DEHP (900 mg/kg/day)-challenged rats showing azospermia in some seminiferous tubules (arrow) (H and E, ×80). (F) Testicular cross sections from rats pretreated with quercetin prior to DEHP (900 mg/kg/day) challenge showing normal intact histological structure of the seminiferous tubules with complete spermatogenic series in the lumen (arrow) (H and E, ×40). H and E: hematoxylin and eosin; DEHP: di-(2-ethylhexyl) phthalate.

Discussion

The significant decrease in relative testis weight observed in this study, after treatment with 900 mg/kg of DEHP, may be explained by the significant decrease in absolute testes weight and unchanging of body weight. The weight of the testis is largely dependent on the mass of the differentiated spermatogenic cells, and the reduction in the weight of the testis may be due to decreased number of germ cells, inhibition of spermatogenesis and steroidogenic enzyme activity.³⁵ The testicular sperm counts and DSP are important indicators of spermatogenesis.³⁶ In this study, administration of DEHP reduced sperm count and motility and DSP. Sperm count and motility correlated with testis weight and plasma testosterone concentration, so that the motility of sperm was only affected in rats treated with 900 mg of DEHP. This might be due to the low testosterone level in these animals. Another explanation is that when sperm production is drastically reduced, fewer sperm pass from the testis to the cauda, resulting in decreased spermatozoa in the cauda epididymis. The observed decrease in the epididymal spermatozoa numbers may therefore indicate that DEHP affects the early stages of spermiogenesis, which is well supported by the observed decrease in daily spermatozoa production. These results come in accordance with³⁷ who showed an obvious reduction in the total sperm count and number of sperm heads after DEHP treatment. Moreover, disturbed testicular histology and spermatogenesis, diminished testosterone, and sperm motility were reported after DEHP treatment.³⁸ Further, Ge et al.³⁹ and Noriega et al.⁹ reported that the mechanisms by which DEHP exerts its toxic effects in reproductive system is related to its anti-androgenic potential. However, pretreatment with quercetin ameliorated the DEHP-induced sperm toxicity by maintaining the sperm quality near normalcy.

The testis is an androgen-dependent organ and testosterone is essential to maintain the structure and function of the testis and accessory sex glands.⁴⁰ It is interesting to note that decreased testosterone secretion would be a result of oxidative stress that is related to insufficiency in the activity of key enzymatic and nonenzymatic antioxidants inside Leydig cells.⁴¹ Available data suggest that Leydig cells are one of the main targets of phthalates.³⁹ Leydig cells are the primary source of testosterone production in males, and differentiation of Leydig cells in the testes is one of the primary events in the development of the male body and fertility.⁴²

ACP is considered to be functional indicator of spermatogenesis. The increased activity of TACP in DEHP-administered animals reflects the release of this phosphatase from the lysosomes of the degenerating cells and rapid catabolism of the injured germ cells.⁴³ The decreased activity of ACP in the prostate might be due to the increased permeability of plasma membrane or cellular necrosis, thereby showing the stress condition of the treated animals.⁴⁴

LDH-X is one of the best characterized germ cell-specific isozyme that plays an important role in the process of spermatogenesis and has been shown to be vital for sperm survival and motility.⁴⁵ LDH-X is the predominant isozyme in the pachytene spermatocyte and round and condensing spermatids, whereas spermatozoa contain only LDH-X.⁴⁶ The significant decrease of LDH-X activity, a consequence of enhanced LPO in DEHP-treated animals, may be due to disintegration of the mitochondrial membrane ultrastructure which in turn affects the membrane-bound LDH-X function. LDH-X plays an important role in transferring hydrogen from cytoplasm to mitochondria by redox-coupling α-hydroxy acid/α-keto acid related to spermatozoal metabolism.⁴⁷ This could be one of the contributory factors leading to reduced male sperm concentration and sperm motility on DEHP exposure.⁴⁸ The reduction in LDH-X activity in rats treated with 900 mg/kg indicates a suppression of essential sperm maturational processes that precede the penetration of the oocyte by the sperm, such as capacitation and acrosome reaction, which can result in decline of fertility.⁴⁹ The alteration in these enzymes activity may lead to destruction of seminiferous epithelium and loss of germinal elements, resulting in reduction in the number of spermatids associated with decrease in the DSP in the testes.⁵⁰ The results obtained in this study indicate that pretreatment with quercetin influences the activities of these enzymes to an appreciable extent and suggests the cytoprotective action of quercetin in preventing DEHP-induced testicular damage.

Oxidative stress has been implicated as a major causative factor in male reproductive dysfunction.⁵¹ Studies showed that there is a correlation between reproductive toxicity induced by environmental contaminants and oxidative stress.⁵² Previous data have also shown that phthalates were able to produce free radicals by several pathways in germ cells, suggesting the possibility that oxidative stress and mitochondrial dysfunction in germ cells may contribute to phthalate-induced disruption of spermatogenesis.^53,54 DEHP treatment, indeed, was reported to provoke oxidative stress as measured by increases in ROS in subsequently isolated rat spermatocytes¹⁰ and in MA-10 mouse Leydig tumor cell line.⁵⁵ Thus, at least one of the mechanisms underlying the reproductive toxicity of DEHP might be the induction of intracellular ROS and/or to cause alterations on intracellular enzymatic and nonenzymatic antioxidants, thereby to produce oxidative stress. The spermatozoa membranes are rich in polyunsaturated fatty acids, so they are susceptible to ROS attack and LPO.⁵⁶ LPO causes membrane damage that leads to a decrease in sperm motility, presumably by a rapid loss of intracellular ATP, and an increase in sperm morphology defects.⁵⁷ Antioxidants play a major role by continuously inactivating ROS to keep only a small amount necessary to maintain normal cell function.⁵⁸ In the present study, the activities of testicular SOD and CAT were significantly decreased in rats treated with 900 mg/kg of DEHP. The existence of a mutually supportive relationship between enzymatic antioxidants, SOD and CAT, against accumulation of ROS inactivates the superoxide anion and peroxide radicals by converting them into water and oxygen. In this study, the observed decrease in SOD activity suggests inactivation of the enzyme possibly due to increased superoxide radical production or an inhibition by the H₂O₂ as a result of corresponding decrease in the activity of CAT, which selectively degrades H₂O₂.⁵⁹ For more explanation, significant depletion of the testicular antioxidant enzymes and overproduced toxic free radicals leads to a more pro-oxidant environment which per se can significantly increase the susceptibility of spermatogenic cells to oxidative stress.⁴⁰ In the present study, DEHP (600 and 900 mg/kg) administration depleted GSH content in testicular tissues that made spermatogenic cells more susceptible to oxidative damage, especially during increased free radical production.⁶⁰ GSH is an important intracellular antioxidant that spontaneously neutralizes several ROS.

Quercetin is reported to ameliorate the carbon tetrachloride induced oxidative toxicity in rat testis.⁶¹ Also, cytotoxicity in Sertoli-germ cell cultures due to atrazine is countered by quercetin intervention.¹⁹ Similarly, oxidative damage induced by diethylstilbestrol to hamster spermatogenic cells is also prevented by simultaneous treatment with quercetin.⁶² Our study showed that quercetin significantly reduced LPO and provided protective effect against testicular oxidative damage in testes of animals treated with DEHP. The testicular toxicity induced by DEHP is further confirmed by the abnormal histologic findings. DEHP-treated rats showed degeneration of spermatogonial cells in some seminiferous tubules (300 mg/kg), degeneration and necrosis with pyknosis in the nuclei of spermatogonial cells and spermatid (600 mg/kg), and azospermia in some seminiferous tubules (900 mg/kg). These results suggest that DEHP could induce injury of spermatogonia and spermatids, and DEHP can decrease spermatogenesis by adversely affecting spermatogonia followed by depletion of spermatids and spermatozoa. Quercetin-pretreated animals showed normal intact histological structure of the seminiferous tubules with complete spermatogenic series in the lumen, thereby highlighting its protective role in countering the testicular toxicity induced by DEHP.

In conclusion, the findings of this study demonstrate that DEHP impairs testicular function at least, in part, by inducing oxidative stress, and quercetin has a potent protective effect against DEHP-induced testicular toxicity in rats.

Footnotes

Acknowledgments

The authors thank Dr Adel M. Bakeer, Prof. of Pathology, Faculty of Veterinary Medicine, Cairo University, for his help in performing the histopathological examination. The authors acknowledge with thanks Faculty of Pharmacy, Al-Azhar University, for technical and financial support.

Conflict of interest

The authors declared no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

References

Muczynski

Lecureuil

Messiaen

. Cellular and molecular effect of MEHP involving LXRα in human fetal testis and ovary. PLoS One 2012; 7: e48266.

Kavlock

Boekelheide

Chapin

. NTP center for the evaluation of risks to human reproduction: phthalates expert panel report on the reproductive and developmental toxicity of di-n-butyl phthalate. Reprod Toxicol 2002; 16: 489–527.

Albert

Jégou

. A critical assessment of the endocrine susceptibility of the human testis to phthalates from fetal life to adulthood. Hum Reprod Update 2014; 20: 231–249.

Pan

Yuan

. Plasticizer contamination in edible vegetable oil in a U.S. retail market. J Agric Food Chem 2013; 61(39): 9502–9509.

Gray

. Testicular toxicity in vitro: sertoli-germ cell co-cultures as a model system. Food Chem Toxicol 1986; 24: 601–605.

Parks

Ostby

Lambright

. The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat. Toxicol Sci 2000; 58: 339–349.

Lin

Chen

. Involvement of testicular growth factors in fetal Leydig cell aggregation after exposure to phthalate in utero. Proc Natl Acad Sci USA 2008; 105: 7218–7222.

Barlow

McIntyre

Foster

. Male reproductive tract lesions at 6, 12, and 18 months of age following in utero exposure to di(n-butyl) phthalate. Toxicol Pathol 2004; 32: 79–90.

Noriega

Howdeshell

Furr

. Pubertal administration of DEHP delays puberty, suppresses testosterone production, and inhibits reproductive tract development in male sprague-dawley and long-evans rats. Toxicol Sci 2009; 111: 163–178.

10.

Kasahara

Sato

Miyoshi

. Role of oxidative stress in germ cell apoptosis induced by di(2-ethylhexyl)phthalate. Biochem J 2002; 365: 849–856.

11.

Latchoumycandane

Mathur

. Effect of methoxychlor on the antioxidant system in mitochondrial and microsome-rich fractions of rat testis. Toxicology 2002; 176: 67–75.

12.

Erkekoglu

Rachidi

De Rosa

. Protective effect of selenium supplementation on the genotoxicity of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl) phthalate treatment in LNCaP cells. Free Radic Biol Med 2010; 49: 559–566.

13.

Erkekoglu

Rachidi

Yuzugullu

. Evaluation of cytotoxicity and oxidative DNA damaging effects of di(2-ethylhexyl)-phthalate (DEHP) and mono(2-ethylhexyl)-phthalate (MEHP) on MA-10 Leydig cells and protection by selenium. Toxicol Appl Pharmacol 2010; 248: 52–62.

14.

Erkekoglu

Giray

Rachidi

. Effects of di(2-ethylhexyl)phthalate on testicular oxidant/antioxidant status in selenium-deficient and selenium-supplemented rats. Environ Toxicol 2014; 29: 98–107.

15.

Ramyaa

Padma

. Ochratoxin-induced toxicity, oxidative stress and apoptosis ameliorated by quercetin-Modulation by Nrf2. Food Chem Toxicol 2013; 62: 205–216.

16.

Aly

Khafagy

. Taurine reverses endosulfan-induced oxidative stress and apoptosis in adult rat testis. Food Chem Toxicol 2014; 64: 1–9.

17.

Anjaneyulu

Chopra

. Quercetin, an anti-oxidant bioflavonoid, attenuates diabetic nephropathy in rats. Clin Exp Pharmacol Physiol 2004; 31: 244–248.

18.

Coskun

Kanter

Armutcu

. Protective effects of quercetin, a flavonoid antioxidant, in absolute ethanol-induced acute gastric ulcer. Eur J Gen Med 2004; 1: 37–42.

19.

Abarikwu

Pant

Farombi

. Quercetin decreases steroidogenic enzyme activity, NF-κB expression, and oxidative stress in cultured Leydig cells exposed to atrazine. Mol Cell Biochem 2013; 373: 19–28.

20.

Kang

Huang

. Exposure to DEHP and MEHP from hatching to adulthood causes reproductive dysfunction and endocrine disruption in marine medaka (Oryzias melastigma). Aquat Toxicol 2013; 146C: 115–126.

21.

Ryu

Whang

Park

. Di(2-ethylhexyl) phthalate induces apoptosis through peroxisome proliferators-activated receptor-gamma and ERK 1/2 activation in testis of Sprague-Dawley rats. J Toxicol Environ Health A 2007; 70: 1296–1303.

22.

Kwack

Han

Park

. Comparison of the short term toxicity of phthalate diesters and monoesters in sprague-dawley male rats. Toxicol Res 2010; 26: 75–82.

23.

Taepongsorat

Tangpraprutgul

Kitana

. Stimulating effects of quercetin on sperm quality and reproductive organs in adult male rats. Asian J Androl 2008; 10: 249–258.

24.

Banchroft

Stevens

Turner

. Theory and practice of histological techniques. 4th ed. New York: Churchill Livingstone, 1996.

25.

Yokoi

Uthus

Nielsen

. Nickel deficiency diminishes sperm quantity and movement in rats. Biol Trace Elem Res 2003; 93: 141–153.

26.

Blazak

Treinen

Juniewicz

. Application of testicular spermhead counts in the assessment of male reproductive toxicity. Meth Toxicol 1993; 3A: 86–94.

27.

Sharpe

Fisher

Millar

. Gestational and lactational exposure of rats to xenoestrogens results in reduced testicular size and sperm production. Environ Health Perspect 1995; 103: 1136–1143.

28.

Chen

Booksteinm

Meldrum

. Diagnosis of the testosterone secreting adrenal adenoma by selective venous catheterization. Fertil Steril 1991; 55: 1202–1203.

29.

Hillmann

. Continuous photometric measurement of prostate acid phosphatase activity. Z Klin Chem Klin Biochem 1971; 9: 273–274.

30.

Meistrich

Trostle

Frapart

. Biosynthesis and localization of lactate dehydrogenase X in pachytene spermatocytes and spermatids of mouse testes. Dev Biol 1977; 60: 428–441.

31.

Mihara

Uchiyama

. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978; 86, 271–278.

32.

Marklund

. Involvement of superoxide anion radical in autooxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 1974; 47: 469–474.

33.

Claiborne

. Catalase activity. In: Greenwald

(ed) Handbook of Methods for Oxygen Radical Research. Florida: CRC Press, pp. 283–284, 1985.

34.

Moron

Depierre

Mannervik

. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim Biophys Acta 1979; 582: 67–78.

35.

Takahashi

Oishi

. Testicular toxicity of dietary 2,2-bis(4-hydroxyphenyl)propane (bisphenol A) in F344 rat. Arch Toxicol 2001; 75: 42–51.

36.

Wang

Zhang

Cui

. Study of effects of lead acetate on reproductive function in male mice. Chin J Ind Med 2004; 17: 237–239.

37.

Yang

. Di-2-ethylhexyl phthalate and its metabolite single-ethylhexyl phthalate affect TGF-beta 1 expression and telomerase activity in the testis of young male rats. Zhonghua Nan Ke Xue 2012; 18: 783–788.

38.

Erkekoglu

Zeybek

Giray

. Reproductive toxicity of di(2-ethylhexyl) phthalate in selenium-supplemented and selenium-deficient rats. Drug Chem Toxicol 2011; 34: 379–389.

39.

Chen

Tanrikut

. Phthalate ester toxicity in leydig cells: developmental timing and dosage considerations. Reprod Toxicol 2007; 23: 366–373.

40.

Rezvanfar

Shahverdi

. Protection of cisplatin-induced spermatotoxicity, DNA damage and chromatin abnormality by selenium nano-particles. Toxicol Appl Pharmacol 2013; 266: 356–365.

41.

Cao

Durrani

Rustum

. Selective modulation of the therapeutic efficacy of anticancer drugs by selenium containing compounds against human tumor xenografts. Clin Cancer Res 2004; 10: 2561–2569.

42.

Zhang

Lin

Liu

. Disruption effects of monophthalate exposures on inter-Sertoli tight junction in a two-compartment culture model. Environ Toxicol 2008; 23: 302–308.

43.

Selvakumar

Prahalathan

Sudharsan

. Protective effect of lipoic acid on cyclophosphamide-induced testicular toxicity. Clin Chim Acta 2006; 367: 114–119.

44.

Yousef

M.I

. Aluminium-induced changes in hemato-biochemical parameters, lipid peroxidation and enzyme activities of male rabbits: protective role of ascorbic acid. Toxicology 2004; 199: 47–57.

45.

Pan

Sharief

Okabe

. Amino acid sequence studies on lactate dehydrogenase C4 isozymes from mouse and rat testes. J Biol Chem 1983; 258: 7005–16.

46.

Kaur

Bansal

. Influence of selenium induced oxidative stress on spermatogenesis and lactate dehydrogenase-X in mice testis. Asian J Androl 2004; 6: 227–232.

47.

Davis

Lin

. Developmental changes in lactate dehydrogenase-X activity in young jaundiced, male rats. Arch Androl 1989; 22: 131–136.

48.

Benoff

Hauser

Marmar

. Cadmium concentration in blood and seminal plasma, correlations with sperm number and motility in three male populations (infertility patients, artificial insemination donors & unselected volunteers. Mol Med 2009; 15: 248–262.

49.

Yan

Chen

Zuo

. Effects of tributyltin on epididymal function and sperm maturation in mice. Environ Toxicol Pharmacol 2009; 28: 19–24.

50.

Hodgen

Sherins

. Enzymes as markers of testicular growth and development in the rat. Endocrinol 1973; 93: 985–989.

51.

Rezvanfar

Sadrkhanlou

Ahmadi

. Protection of cyclophosphamide-induced toxicity in reproductive tract histology, sperm characteristics, and DNA damage by an herbal source; evidence for role of free-radical toxic stress. Hum Exp Toxicol 2008; 27: 901–910.

52.

Abarikwu

Adesiyan

Oyejola

. Changes in sperm characteristics and induction of oxidative stress in the testis and epididymis of experimental rats by a herbicides, atrazine. Arch Environ Contam Toxicol 2010; 58: 874–882.

53.

Gazouli

Yao

Boujrad

. Effect of peroxisome proliferators on Leydig cellperipheral-type benzodiazepine receptor gene expression, hormone-stimulated cholesterol transport, and steroidogenesis: role of the peroxisome proliferator-activator receptor alpha. Endocrinology 2002; 143: 2571–2583.

54.

Suna

Yamaguchi

Kimura

. Preventive effect of dpsicose, one of rare ketohexoses, on di-(2-ethylhexyl) phthalate (DEHP)-induced testicular injury in rat. Toxicol Lett 2007; 173: 107–117.

55.

Fan

Traore

. Molecular mechanisms mediating the effect of mono-(2-ethylhexyl) phthalate on hormone-stimulated steroidogenesis in MA-10 mouse tumor Leydig cells. Endocrinology 2010; 151: 3348–3362.

56.

Mandava

Rao

. Antioxidative potential of melatonin against mercury induced intoxication in spermatozoa in vitro. Toxicol In Vitro 2008; 22: 935–942.

57.

Guthrie

Welch

. Effects of reactive oxygen species on sperm function. Theriogenology 2012; 78: 1700–1708.

58.

Oyagbemi

Adedara

Saba

. Role of oxidative stress in reproductive toxicity induced by co-administration of chloramphenicol and multivitamin-haematinics complex in rats. Basic Clin Pharmacol Toxicol 2010; 107: 703–708.

59.

Adaramoye

Adedara

Farombi

. Possible ameliorative effects of kolaviron against reproductive toxicity in sub-lethally whole body gamma-irradiated rats. Exp Toxicol Pathol 2012; 64: 379–385.

60.

El-Desoky

Bashandy

Alhazza

. Improvement of mercuric chloride-induced testis injuries and sperm quality deteriorations by Spirulina platensis in rats. PLoS One 2013; 8: e59177.

61.

Sönmez

Türk

Ceribasi

. Quercetin attenuates carbon tetrachloride-induced testicular damage in rats. Andrologia 2013; 46(8): 848–858.

62.

Shi

. Quercetin protects hamster spermatogenic cells from oxidative damage induced by diethylstilboestrol. Andrologia 2010; 42: 285–290.

Quercetin attenuates di-(2-ethylhexyl) phthalate-induced testicular toxicity in adult rats

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Animals and treatment

Necropsy

Sperm count and motility

Daily sperm production

Serum testosterone

Serum TACP and PACP activity

Testicular LDH-X

Testicular oxidative stress status

Statistical analysis

Results

Relative testes weight and sperm parameters

Serum testosterone and ACP (total and prostatic)

Testicular LDH-X

Oxidative stress status

Histopathological examination

Discussion

Footnotes

Acknowledgments

Conflict of interest

Funding

References