Andrology & Gynecology: Current ResearchISSN: 2327-4360

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Research Article, Androl Gynecol Curr Res Vol: 4 Issue: 3

Lindane Induces Spermatotoxicity and inhibits Steroidogenesis in Adult Rats

Hamdy AA Aly1,2*, Abdulrahman M Alahdal3, Alaa Bagalagel3 Ahmed M Mansour2, Abdel-Moneim M Osman4, Ibrahim A Shehata5, and Waleed El-Shaer 6
1Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
2Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt
3Department of Clinical Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
4Department of Pharmacology, College of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
5Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
6Departments of urology, Banha University Hospital, Egypt
Corresponding author : Hamdy AA Aly
Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
Tel:
+966 0542497023
E-mail: [email protected]
Received: January 13, 2016 Accepted: October 25, 2016 Published: November 01, 2016
Citation: Hamdy AA Aly, Abdulrahman M. Alahdal, Alaa Bagalagel, Mansour AM, Osman AM, Shehata IA, El-Shaer W (2016) Lindane Induces Spermatotoxicity and Inhibits Steroidogenesis in Adult Rats. Androl Gynecol: Curr Res 4:2. doi: 10.4172/2327-4360.1000150

Abstract

This study was conducted to investigate the influence of lindane on spermatogenesis and testicular biochemical parameters and to get insight into its mechanism. Adult male albino rats were treated orally with lindane at doses of 0, 1, 2 or 4 mg/kg/day for 30 consecutive days. Testes weight and sperm count and motility were significantly decreased. Testicular activities of 3β-HSD, 17β-HSD were significantly inhibited. Testicular cholesterol level was significantly increased while glycogen and sialic acid content were significantly decreased. Testicular activities of LDH-X, γ-GT, β-glucuronidase and acid phosphatase (ACP) were significantly decreased. Aldo-ketoreductase activity was significantly decreased in response to 2 or 4 mg/kg/day of lindane while, protein carbonyl contentswere significantly increased. Hydrogen peroxide and hydroxyl radical generation and LPO were significantly increased. The enzymatic antioxidants SOD, CAT and GPx and the non-enzymatic antioxidants GSH and Vit C were significantly decreased. Lindane at doses 2 and 4 mg/kg/day showed a significant reduction in Vit E, while at a dose of 1 mg/kg/day did not show any significant change. In conclusion, lindane inhibits spermatogenesis, steroidogenesis and suppresses Sertoli cell marker enzymes which may be due, partly, to oxidative stress. Lindane enhances ROS generation and LPO and depletes antioxidant enzymes and non-enzymatic antioxidants.

Keywords: Lindane; Testis; Spermatogenesis; Steroidogenesis; Oxidative stres

Keywords

Lindane; Testis; Spermatogenesis; Steroidogenesis; Oxidative stress

Introduction

Exposure to toxins during spermatogenesis may target male germ cells that results in abnormal functioning and adverse pregnancy outcomes [1]. In the past few years, there has been increased interest in assessing the relationship between impaired male fertility and environmental factors [2,3]. Lindane, the 6-isomer of hexachlorocyclohexane (also known as -HCH), is an organ chlorinated insecticide, widely used in agriculture and as a therapeutic scabicide, pediculicide, and ectoparasiticide for humans and animals [4]. The extensive use, chemical stability and bioaccumulation potential of lindane have resulted in its ubiquitous distribution in the ecosystem and thereby considered to be a global pollutant [5].
Several studies have demonstrated the repro-toxic effects of lindane in rodents and in fact, male gonads have been found to be highly sensitive target organs for lindane [6]. Degenerative changes such as tubular atrophy, necrosed spermatogenic cells, and enlargement of the interstitial space have been observed in adult rat testis following oral (17.6 mg/kg for 90 days) administration of lindane [7]. Other reported reproductive effects of lindane include alteration in activities of ATPase of testicular plasma membrane [8], impaired spermatogenesis [9] and steroidogenesis [10] decreased sperm count [11] and increased sperm abnormalities [12], changes in testosterone metabolism and plasma testosterone levels [12,13], and alterations in Leydig and Sertoli cells [6,14]. All of these effects impair male reproductive function. Lindane has been reported to induce oxidative stress by interacting with the cell membrane, triggering the generation of ROS and altering the level of antioxidant molecules which in turn cause severe physiological dysfunction in various organs such as liver, testes and brain [15,16]. Alterations in the levels of heat shock protein and clusterin accompanied by an induction of stress in rat testis as early as 12 h following exposure to lindane were also reported [17].
Transient inhibitory effect of lindane on testicular steroidogenesis and the possible role of hydrogen peroxide (H2O2) in mediating these effects were demonstrated by Saradha [18]. Other studies clearly demonstrated the detrimental effects of lindane on testicular functions and its strong association with oxidative stress and reactive oxygen species (ROS) [19]. The stress response and programmed cell death are cellular reactions to stressful stimuli. Stress-induced apoptotic alterations have been implicated as a cause or consequence of various pathological states including infertility [20,21]. Still through studies on testicular toxicity by lindane are lacking which needs attention in greater detail. This study was conducted to further investigate the influence of lindane on spermatogenesis and testicular biochemical parameters in order to assess the male reproductive toxicity of lindane and to get more insight into its mechanism.

Materials and Methods

Chemicals
Lindane (γ-isomer) (99% pure), pyrogallol, sodium azide, Alexa Fluor-488-PNA, Rh123 and glutathione reductase were purchased from Sigma–Aldrich Chemical Company, St. Louis, MO, USA. All other chemicals are of analytical grade.
Animals and treatments
Healthy adult male Wistar rats (90 days) weighing 180-200 g were housed in clean polypropylene cages and maintained on a 12 h light: dark cycle and a temperature of 20-25°C with ad libitum access to food (stand ard rat chow) and water. All the experiments with animals were carried out according to the guidelines of the Institutional Animal Ethical Committee. For 7 days before the experiment, rats were handled daily for 5 min to acclimatize them to human contact and minimize their physiological responses to hand ling for subsequent protocols [22]. Lindane was dissolved in olive oil and given to rats by gavage at 0, 1, 2 or 4 mg/kg/day for 30 consecutive days. Gavage volume was adjusted according to the weight of each rat. The doses and duration were selected based on as per previous publication [23].
Necropsy
Twenty four hours after the last dose, the animals were euthanized under ether anesthesia; testes were removed, cleaned from adhering fat and connective tissues and weighed. The testes were homogenized in ice-cold phosphate buffer (pH 7.0) using a glass-teflon homogenizer. The homogenate was centrifuged at 10,000 × g for 30 min at 4°C and the supernatant used for other biochemical and enzymes estimation as enzyme source. Protein concentrations were determined using a BCA kit (Pierce, Rockford, USA) that employed bovine serum albumin as a standard. The cauda epididymides from each animal were used for sperm count and motility.
Sperm count and motility
Cauda epididymides were dissected out, weighed, 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 sperms was recorded using a phase contrast microscope at a magnification of 400 x. Total sperm number was determined by using a Neubauer hemocytometer as previously described [24]. To determine sperm motility, 100 sperms each were observed in 3 different fields, and classified into motile and non-motile sperms, and the motility was expressed as percentage incidence. Aliquots of this sperm suspension were used to assess the acrosomal integrity, mitochondrial membrane potential (Δψm) and 5’-nucleotidase activity of sperm and the others were used for following biochemical studies. In the same manner, caput/corpus portion of the epididymis was cut into mall fragments with scissors, and sperm counted as described for the cauda epididymis.
Testicular steroidogenesis enzyme activities
The testicular tissue was homogenized in ice-cold phosphate buffer (pH 7.0) using a glass-teflon homogenizer. The activities of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) were measured by the method of Bergmeyer [25]. The reaction mixture in a volume of 2.0 ml contained 100 μmol of sodium pyrophosphate buffer (pH 9.0) and 0.5 μmol cofactor NAD for 3β-HSD and NADPH for 17β-HSD, 0.08 μmol of substrate (dehydroepiand rosterone for 3β-HSD and and rostenedione for 17β-HSD) and 100 μl of testicular protein. The reactions were carried out in a quartz cuvette of 1.0 cm path length at 23 ± 1°C. The absorbance at 340 nm was measured at 20 s intervals for 3 min in a UV-Vis spectrophotometer (UV-1700 Shimadzu, Japan). The enzyme activities were expressed as nmol of NAD converted to NADH/min/mg protein (3β-HSD) or nmol of NADPH converted to NADP/min/mg protein (17β-HSD).
Glycogen and cholesterol
Glycogen was assayed by the method of Montgommery [26]. Briefly the assay mixture contained 30% KOH, H2SO4, 80% Phenol and absolute alcohol. Polysaccharides are treated with conc. H2SO4 and phenol due to which they undergo degradation and form a complex which is pink in colour. The intensity of this colour indicates intensity of glycogen in the tissue. Total cholesterol was determined by the method of Zlatkis [27]. Briefly it contains FeCl3 solution, conc. H2SO4 and glacial acetic acid. The phenanthrene ring of cholesterol reacts with FeCl3.7H2O and gives pinkish to brown colour depending upon the concentration of cholesterol.
Sialic acid
Sialic acid content was estimated by using the method of Aminoff [28]. Briefly, 500 μl of the enzyme source was treated with 250 μl periodate reagent and incubated at 37°C in a water bath for 30 min. Then, the excess of periodate was reduced by sodium arsenite (2% sodium arsenite in 0.5 N HCl). After 5 min, as soon as the yellow color of liberated iodine started fading off, 2 ml of thiobarbituric acid (pH 9.0) was added and with a stopper, the tubes were kept in boiling water bath for 8 min. The tubes were cooled in an ice water and shaken with butanol mixture (butan-1-ol containing 5% 12 N HCl), the separation of two phases was done by a short rapid centrifugation, and color intensity in the butanol phase was measured at 549 nm. The values are expressed as μg/mg protein.
Marker enzymes
Lactate dehydrogenase-X (LDH-X) activity was measured using a-ketovaleric acid as the substrate [29]. γ-Glutamyl transferase (γ- GT) was measured by the method of Orlowski and Meister [30]. Assessment of β-glucuronidase was carried out using p-nitrophenyl- β-D-glucuronide [31]. Acid phosphatase (ACP) activity was measured following the methodology of assay kit supplier (Acid phosphatase diagnosis kit). Aldo-keto reductase activity was determined as described previously [32]. The rate of decrease in the absorbance of NADPH at 340 nm was measured at room temperature with methyl glyoxal (MG), a common substrate for the aldo-ketoreductase family, as the substrate.
Determination of protein carbonyl content
Protein carbonyl content was determined by the most common and reliable method based on the reaction of carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) to form a 2,4- dinitrophenyl hydrazone [33]. Briefly, 100 μl of testis homogenate was incubated with 0.5 ml DNPH for 60 min. Subsequently, the protein was precipitated from the solution with the use of 20% trichloroacetic acid. The pellet was washed after centrifugation (3400 × g) with ethyl acetate: ethanol (1:1 v/v) mixture thrice to remove excess of DNPH. The final protein pellet was dissolved in 1.5 ml of 6 M guanidine hydrochloride. The carbonyl content was evaluated in a spectrophotometer at 370 nm.
Reactive oxygen species
Hydrogen peroxide (H2O2) production was quantified by the method of Holland and Storey [34]. In brief, 0.1ml testes extract was added to an assay mixture containing KCl (1.13 M), 0.1 ml potassium phosphate (150 mM), 0.05 ml MgCl2 (60 mM), 0.05 ml EDTA (8 mM), 0.1 ml Tris–HCl (200 mM, pH 7.4) and 0.1 ml acetylated ferrocytochrome c (1 mM). The oxidation of ferrocytochrome c, which provides a measure of H2O2 production, was then evaluated at 550 nm, in a spectrophotometer. The H2O2 content of the sample was expressed as μmol/min/mg protein.
Hydroxyl radical production was quantified by the method of Puntarulo and Cedubaum [35]. To 0.1 ml cell extract, 0.2 ml 1 M phosphate buffer, 0.1 ml each magnesium chloride (0.1 M), sodium azide (10 mM), DMSO and NADPH (4 mM) were added and incubated for 10 min at 37 °C. The reaction was arrested by adding 0.5 ml chromo tropic acid, boiled for 30 min, and read at 570 nm against a reagent blank. Hydroxyl radical was expressed as μmol/min/ mg protein.
Lipid peroxidation (LPO)
Lipid peroxidation (LPO) was determined by the method of Hogberg [36]. Malondialdehyde (MDA), formed as an end product of the peroxidation of lipids, served as an index of the intensity of oxidative stress. MDA reacts with thiobarbituric acid to generate a color product that can be measured optically at 532 nm.
Enzymatic antioxidants
Superoxide dismutase (SOD) was assayed according to the method of Marklund and Marklund [37]. The unit of enzyme activity is defined as the enzyme required for 50% inhibition of pyrogallol auto-oxidation. The activity of catalase (CAT) was assayed by the method of Sinha [38]. In this method, dichromate in acetic acid is reduced to chromic acetate when heated in the presence of hydrogen peroxide (H2O2), with the formation of perchloric acid as an unstable intermediate. The chromic acetate thus produced is measured colorimetrically at 610 nm. Glutathione peroxidase (GPx) was assayed by the method of Rotruck [39], which is based on the reaction between glutathione remaining after the action of GPx and 5,5’-dithiobis-(2-nitrobenzoic acid) to give a compound that absorbs light at 412 nm.
Non-enzymatic antioxidants
Total reduced glutathione (GSH) was estimated by the method of Moron [40], where the colour developed was read at 412 nm. Ascorbic acid was assayed by the method of Omaye [41]. Ascorbic acid (vitamin C, Vit C) was oxidized by copper to form dehydroascorbic acid and diketoglutaric acid, which were treated with DNPH to form the derivative of bis-2,4-dinitrophenyl hydrazine. This compound in strong sulphuric acid undergoes a rearrangement to form a product, which was measured at 520 nm. A mildly reducing medium with thiourea was used to prevent non-ascorbic chromogen interference. α- Tocopherol (vitamin E, Vit E) was estimated by the method of Desai [42].
Statistical analysis
Differences between obtained values (mean ± SD, n=6) were compared by one way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparison test. A P value less than 0.05 were taken as a criterion for a statistically significant difference.

Results

Testes weight and sperm characteristics
Animals treated with 1, 2 or 4 mg/kg/day of lindane showed significant decrease (p˂0.05, p˂0.01, and p˂0.001 respectively) in absolute testes weight in a dose related manner as compared to the corresponding control (Table 1). Sperm count (10.62%, 20.35% and 24.48% respectively) and motility (7.33%, 11.66% and 16.35% respectively) were significantly decreased in response to lindane (1, 2 or 4 mg/kg/day respectively) as compared to the corresponding control (Table 1).
Table 1: Effect of lindane on testes weights and sperm characteristics
Steroidogenic enzymes
Lindane treatment (1, 2 or 4 mg/kg/day) significantly decreased testicular 3β-HSD (10.3%, 20.57% and 25.11% respectively) and 17β-HSD activities (10.11%, 22.04% and 44.04% respectively) as compared to the corresponding control (Figure 1 A and B respectively).
Figure 1: Effect of lindane on testicular steroidogeneic enzymes Values are expressed as mean ± SD, n = 6. The symbol represents statistical significance (ANOVA) from control: *, P < 0.05. **, P < 0.01. ***, P < 0.001.
Cholesterol and glycogen
Figure 2 (A and B respectively) compares the testicular levels of cholesterol and glycogen in control and lindane-treated rats. Cholesterol was significantly decreased (8%, 11.78% and 18.13% respectively) in a dose related pattern in response to lindane treatment (1, 2 or 4 mg/kg/day) as compared to the corresponding control. Glycogen levels were significantly decreased (16.29%, 20.2% and 24.1% respectively) in response to lindane treatment (1, 2 or 4 mg/kg/day) as compared to the corresponding control.
Figure 2: Effect of lindane on testicular cholesterol and glycogen: Values are expressed as mean ± SD, n = 6. The symbol represents statistical significance (ANOVA) from control: *, P < 0.05. **, P < 0.01, ***, P < 0.001.
Sialic acid and LDH-X
Figure 3 (A and B respectively) shows the effect of lindane treatment on testicular sialic acid and LDH-X. Rats treated with 1, 2 or 4 mg/kg/day of lindane significantly decreased sialic acid content (14.69%, 20.7% and 22.12% respectively) and LDH-X activity (13.64%, 20.91% and 40% respectively) as compared to the corresponding control.
Figure 3: Effect of lindane on testicular sialic acid content and LDH-X activity. Values are expressed as mean ± SD, n = 6. The symbol represents statistical significance (ANOVA) from control: *, P < 0.05. . **, P < 0.01, ***, P < 0.001.
γ-Glutamyl transferase (γ-GT) and β-glucuronidase
The activities of testicular γ-GT (11.01%, 13.76% and 18.8% respectively) and β-glucuronidase (14.15%, 21.63% and 35.83% respectively) were significantly decreased in response to lindane (1, 2 or 4 mg/kg/day) treatment in a dose-related manner as compared to the corresponding control (Figure 4 A and B respectively).
Figure 4: Effect of lindane on testicular γ-Glutamyl transferase (γ-GT) and β-glucuronidase activities. Values are expressed as mean ± SD, n = 6. The symbol represents statistical significance (ANOVA) from control: *, P < 0.05. **, P < 0.01, ***, P < 0.001.
Acid phosphatase (ACP) and aldo-ketoreductase
Animals treated with lindane (1, 2 or 4 mg/kg/day) showed a significant decreased in ACP activity (21.87%, 28% and 38.67% respectively) in a dose related pattern as compared to the corresponding control (Figure 5A). Aldo-ketoreductase enzyme demonstrated a significant decrease (29.03% and 32.38% respectively) in response to lindane (2 or 4 mg/kg/day) treatment, while 1 mg/kg/ day of lindane did not show any significant change as compared to the corresponding control (Figure 5B).
Figure 5: Effect of lindane on testicular acid phosphatase (ACP) and aldo-keto reductase activities. Values are expressed as mean ± SD, n = 6. The symbol represents statistical significance (ANOVA) from control: *, P < 0.05**, P < 0.01.
Protein carbonyl contents
Figure 6 displays the change in carbonyl contents in response to lindane treatment. Carbonyl contents were significantly increased (22.47%, 24.34% and 30.34% respectively) in response to lindane (1, 2 or 4 mg/kg/day) treatment as compared to the corresponding control.
Figure 6: Effect of lindane on testicular protein carbonyl content. Values are expressed as mean ± SD, n = 6. The symbol represents statistical significance (ANOVA) from control: *, P < 0.05. **, P < 0.01.
Reactive oxygen species and lipid peroxidation
The effect of lindane treatment on reactive oxygen species such as hydrogen peroxide (H2O2) and hydroxyl radical production in rat testis are presented in Figure 6. Lindane (1, 2 or 4 mg/kg/ day) treatment significantly increased H2O2 (23.26%, 36.28% and 40.93% respectively) and hydroxyl radical (24.55%, 30.54% and 35.93% respectively) production as compared to the corresponding control. Lipid peroxidation in rat testis was significantly increased (19.09%, 26.36% and 28.64% respectively) in response to lindane (1, 2 or 4 mg/kg/day) as compared to the corresponding control.
Enzymatic and non-enzymatic antioxidants
The activities of the testicular enzymatic antioxidants SOD (15.8%, 30.04% and 46.62% respectively), CAT (9.84%, 12.35% and 15.97% respectively) and GPx (15.95%, 20.23% and 32.48% respectively) were significantly decreased in response to lindane (1, 2 or 4 mg/ kg/day) treatment as compared to the corresponding control (Table 2). The levels of the non-enzymatic antioxidants GSH (16.67%, 23.81% and 25.43% respectively) and Vit C (14.44%, 20.86% and 21.39% respectively) were significantly decreased in response to 1, 2 or 4 mg/kg/day of lindane as compared to the corresponding control (Table 2). Animals treated with 2 or 4 mg/kg/day of lindane showed a significant decreased in Vit E level (16.92% and 19.49% respectively) as compared to the corresponding control. Lindane at a dose of 1 mg/kg/day did not show any significant change in Vit E level (Table 2).
Table 2: Effect of lindane on enzymatic and non-enzymatic antioxidants

Discussion

The current study was conducted to interpret the testicular response to lindane in adult male rats. Because the weight of the test is largely depends on the mass of the differentiated spermatogenic cells, a reduction in the organ weight may be attributed to decreased sperm production [43]. The decrease in the sperm count in the present study may be due to the decreased levels of intratesticular testosterone, as testosterone level is directly linked to spermatogenesis [44]. It is also possible that the Sertoli cells might have been affected and the other possibility might be due its effect on the epididymal function. Sperm motility assessment is an integral part of some reproductive toxicity test guidelines [45]. It is known that pesticides may reduce mitochondrial enzyme activity of spermatozoa [46], which will result in a reduction of sperm motility. To delineate the effect of lindane on steroidogenesis, the activities of 3β-HSD and 17β-HSD enzymes were assayed. 3β-HSD and 17β-HSD are the key enzymes responsible for the regulation of testosterone biosynthesis [47]. This inhibition in testicular 3β-HSD and 17β -HSD activities may be due to membrane lipid peroxidation and could lead to decrease in testosterone production [48]. An increase in oxidative stress causes ROS-induced damage to macromolecules such as DNA, protein, and key enzymes involved in testicular steroidogenesis and spermatogenesis [49]. Steroid hormones are made from cholesterol and the rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone by the mitochondrial cholesterol side chain cleavage enzymes [50]. Increased accumulation of the testicular cholesterol suggests that it is not used in the testosterone biosynthesis, which corroborates that lindane inhibits steroidogenesis [51]. Increased level of cholesterol in testes is attributed to decreased and rogen concentration [52].
Significantly decreased glycogen content may be due to the inhibition of glycolysis during spermatogenesis [53]. The developing spermatozoa consume glucose as well as fructose as energy source [54]. Inhibition of glycogen synthesis eventually decreased spermatogenesis [55]. A significant depletion in sialic acid contents of testes indicates reduced androgen supply to the testes and decrease in number of spermatozoa in lumen [56]. It is possible that significantly low levels of sialic acid in the present study after lindane treatment could have had an impact on spermatogenic processes as evidenced by the decrease in cauda epididymal sperm count [57].
In the present study, activities of marker enzymes like, LDH-X, γ-GT, β-glucuronidase, acid phosphatase (ACP) and aldo-ketoreductase are considered to be functional indicators of spermatogenesis. The decreased level of LDH-X may be due to increased lipid peroxidation in the mitochondria of rat testis. Since the activity of LDH-X is closely associated with spermatogenesis and male testicular development, the decreased activity of this enzyme represents a defect in spermatogenesis and testicular maturation. Toxicants which have direct effect on Sertoli cell function appear to be involved in the control of spermiation, and when disturbed caused epithelial disorganization and subsequent tubular atrophy [58]. γ-GT, a membrane-bound enzyme, is the marker of Sertoli cell function and its activity parallels with the pattern of Sertoli cell maturation and replication [59]. γ-GT is a membrane-bound enzyme involved in the transport and metabolism of glutamyl residues derived from the amino acid glutamic acid. It is involved in GSH metabolism, degrades extracellular GSH to glutamine and cysteinyl glycine, in which cysteine and glycine are absorbed into the cell for intracellular GSH synthesis [60]. Reduced activity of γ-GT leads to decreased intracellular GSH content as observed in this study [61]. The marked decrease in the activity of γ-GT in lindane-treated rats indicates a disruption of the regulatory mechanism resulting in uncontrolled cell metabolism, impaired functions of Sertoli cells [62]. β-Glucuronidase, an enzyme that catalyzes the hydrolysis of β-glucuronic acid, is present in lysosomes of Sertoli cells from rat testis and considered as an important marker of Sertoli cell function [63]. The deleterious effect of lindane on Sertoli cells is further supported by decrease in β-glucuronidase activity, thereby indicating a disruption of the regulatory mechanism resulting in uncontrolled cell metabolism and altered cell function [64]. This finding indicates that γ-GT and β-glucuronidase in Sertoli cells are targeted by lindane. Further, assessment of ACP activity showed substantial decrease in a dose related manner. The decreased activity of acid phosphatase reflects the release of the enzyme from the lysosomes of the degenerating cells and rapid catabolism of the injured germ cells. Acid phosphatase has been used as a marker of testicular toxicity [65] and it is related to Sertoli cell function [66]. The alteration in ACP activity may lead to the destruction of seminiferous epithelium and loss of germinal elements, resulting in the reduction of the number of spermatids associated with the decrease in the daily sperm production in the testes [67].
The aldo-ketoreductases (AR) represent a growing oxidoreductase superfamily that reduces carbonyl groups to alcohols using NADPH as the electron donor [68]. ARs are present at high levels in Sertoli cells and in elongated spermatids and other spermatogenic cells [69]. Sertoli cells which are enriched in ARs would function to detoxify various carbonyl-containing metabolic products [70]. Inhibition of ARs may impair the function of male reproductive tissues by permitting toxic carbonyl compounds to accumulate [69]. It is well known that proteins are highly susceptible to damage by ROS. The reaction of free radicals with the side chains of amino acid residues present in the protein leads to the formation of carbonyl derivatives. Oxidative damage of proteins may lead to the structural alteration and functional inactivation of many enzymes and receptor proteins involved in cell signaling [71]. The increased levels of protein carbonyls in lindane-induced testicular tissue in the present findings might be due to the impaired endogenous antioxidant system and depletion in GSH levels or reduced aldo-keto reductase activity [43].
The present work also shows that the increased H2O2 and hydroxyl radical’s production and lipid peroxidation are accompanied by concomitant decrease in the activities of antioxidant enzymes namely SOD, CAT and GPX as well as the levels of GSH, Vit C and Vit E. Excessive ROS generation leading to lipid peroxidation, oxidative stress and excessive damage of cellular macromolecules (protein, lipids and nucleic acids) has been shown to be a major contributor to the toxicity of contaminants [72]. ROS can cause tissue damage leading to impaired cellular function, alterations in the physiochemical properties of cellular membranes, apoptosis and reduced enzyme/protein activity [73]. Indeed, it has been shown that free radicals inhibit steroidogenesis by interfering with cholesterol transport to mitochondria and /or catalytic function of P450 enzymes [74]. ROS can damage critical components of the steroidogenic pathway in Leydig cells [75]. LPO is one of the main manifestations of oxidative damage initiated by ROS and it has been linked to the altered membrane structure and enzyme inactivation. The increase in LPO reported here, may be the result of increased production of free radicals and /or a decrease in antioxidant status. The sperm cell membrane contains high levels of polyunsaturated fatty acids that bestows considerable fluidity, necessary to allow the cell to fuse with the oocyte [76]. This, however, renders the cells more susceptible to the generation of lipid peroxides. LPO in spermatozoa results in decreased membrane fluidity and a reduced capacity for fertilization. The decrease in activities of the antioxidant enzymes might predispose the sperms to increased free radical damage, because SOD can catalyze decomposition of superoxide radicals to produce H2O2. GPX and CAT have been considered the primary scavengers of H2O2 [77]. In the absence of adequate CAT or GPX activity to degrade H2O2, more H2O2 could be converted to toxic hydroxyl radicals and may contribute to the oxidative stress of lindane toxicity. Decline in the activities of these antioxidant enzymes may be due to their inactivation caused by excess ROS production [78]. Thus, the balance of this enzyme system is essential to dispose the superoxide anion and peroxides generated in the sperms. The reduction in the activities of these enzymes and increase in LPO could reflect the adverse effects of lindane on this finely balanced antioxidant system in rat testis. GSH is the major cellular sulfhydryl compound that serves as both a nucleophile and an effective reductant by interacting with numerous electrophilic and oxidizing compounds. It can act as a non-enzymic antioxidant by direct interaction of SH group with ROS, or it can be involved in the enzymatic detoxification reaction for ROS, as a cofactor or coenzyme. Decreased GSH content can explain in addition a decreased concentration of Vit C, which enters the cell mainly in the oxidized form where it is reduced by GSH. Vit C is a first line antioxidative defense in water-soluble compartment. The diminution of this vitamin has serious consequences as, in addition to its antioxidant function, it also plays a role in regenerating other antioxidants [79]. Vit E is the major lipid-soluble chain-breaking antioxidant present in the rat testicular mitochondria and epididymal spermatozoa that protects unsaturated fatty acids in the membranes from peroxidation [80]. It is stated that lipid peroxyl radicals react more rapidly with vitamin E than membrane lipid [80]. The protective mechanism of vitamin E is probably through its capacity to quickly and effectively scavenge lipid peroxyl radicals before their attack on membrane lipid [82]. The decrease in Vit E and Vit C clearly explains the overproduction of ROS and other free radicals.
When taken together, the present data demonstrates inhibitory effect of lindane on sperm count and motility and testicular steroidogenesis and the possible role of ROS in mediating these effects. In addition, lindane impairs Sertoli cell function as evidenced by decreased testicular LDH-X, γ-GT and β-glucuronidase activities. Moreover, lindane increased cholesterol level and decreased glycogen and sialic acid content in the testis. Furthermore, it depletes the antioxidant enzymes SOD, CAT and GPx and the nonenzymatic antioxidants Vit C and E. In conclusion, lindane inhibits spermatogenesis, steroidogenesis and suppresses Sertoli cell marker enzymes which may be due, partly, to oxidative stress. Lindane enhances ROS generation and LPO and depletes antioxidant enzymes and non-enzymatic antioxidants. These data provide insight into the mode of action of lindane-induced toxicity in the rat testis.

Conflict of interest statement

The authors report no declarations of interest

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