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Modulatory role of lipoic acid on lipopolysaccharide-induced oxidative stress in adult rat Sertoli cells in vitro
Chemico-Biological Interactions 182 (2009) 112–118
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Modulatory role of lipoic acid on lipopolysaccharide-induced oxidative stress in adult rat Sertoli cells in vitro Hamdy A.A. Aly a,b , David A. Lightfoot c , Hany A. El-Shemy d,∗ a
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt Unit of Cellular and Molecular Pharmacology, Catholic University of Louvain, B-1200 Brussels, Belgium Genomics Core Facility, Department of Plant Soil and Agricultural Systems, SIUC, Carbondale, IL, USA d Faculty of Agriculture Research Park (FARP) and Department of Biochemistry, Faculty of Agriculture, Cairo University, 12513 Giza, Egypt b c
a r t i c l e
i n f o
Article history: Received 25 June 2009 Received in revised form 14 August 2009 Accepted 17 August 2009 Available online 21 August 2009 Keywords: Lipoic acid Sertoli cells Inflammation LPS Testicular toxicity Lipid peroxidation Antioxidants
a b s t r a c t Inflammatory reactions to microbial infections may cause male infertility. The mechanisms of inhibition of spermatogenesis can be studied in vitro using rat Sertoli cells. Bacterial lipopolysaccharides (LPS) induce acute inflammations. So LPS treated Sertoli cells can be used to test for new therapeutic compounds. The present study aimed to investigate the protective efficacy of dl-␣-lipoic acid (LA) on lipopolysaccharide (LPS)-induced oxidative stress in adult rat Sertoli cells. Sertoli cells were divided into 4 groups. Group I served as a control incubated with water (vehicle). Groups II and IV were incubated with 100 M LA for 24 h before incubating Groups III and IV with 50 g/ml lipopolysaccharide (LPS) for 12 h. In Group III cells (LPS-treated, no LA) the lactate concentration was decreased whereas hydrogen peroxide production and lipid peroxidation were significantly increased. Moreover, the activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, catalase, glutathioneS-transferase, glutathione reductase were reduced. The concentrations of antioxidant molecules such as reduced glutathione and vitamin C were significantly decreased. The activities of enzymes normally elevated in Sertoli cells, ␥-glutamyl transpeptidase and -glucuronidase, were significantly decreased. Treatment with LA (100 M) for 24 h before LPS-treatment (Group IV), prevented these changes in enzyme activities and metabolite concentrations. Therefore, LA may have a cyto-protective role during LPS-induced inflammation in adult rat Sertoli cells. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction There is a considerable body of clinical evidence suggesting that testis function is decreased during illness or infection, resulting in a temporary or permanent impairment of fertility [1,2]. This impairment is manifested as a decrease in both serum testosterone concentration and sperm counts. The precise mechanism underlying this inhibition is not clearly understood. Of the many causes of male infertility, oxidative stress (OS), a condition mediated by reactive oxygen species (ROS), has been attributed to affect the fertility status. Consequently, OS has been studied extensively in recent years. Evidence now suggests that ROS is a significant contributing pathology in 30–80% of male infertility cases [3–5]. The Sertoli cells play a major role in regulation of spermatogenesis and altering rates of spermatozoa produced. Functions include; providing structural support and nutrition to developing germ cells; phagocytosis of degenerating germ cells and residual bodies; release of spermatids at spermiation; and production of a
∗ Corresponding author. E-mail address: [email protected] (H.A. El-Shemy). 0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2009.08.013
host of proteins that regulate and/or respond to pituitary hormone release; and influence mitotic activity of spermatogonia [6,7]. Tight junctions between adjacent Sertoli cells create the blood–testis barrier, which is the barrier between germ cells situated within the basal and adluminal compartments [8,9]. Protection of developing germ cells, mediated by Sertoli cells, from harmful influences has a high priority in all species [10]. Sertoli cells have long been known to be the targets for various toxicants and infectious agents [11] and a well-established model for toxicity testing in male reproductive systems, both in vivo and in vitro [12]. -Glucuronidase (EC 3.2.1.31) and ␥-glutamyl transpeptidase (␥-GT; 2.3.2.2) activities are considered as important markers of Sertoli cell function [13]. Mature germ cells depend critically on a supply of lactate by Sertoli cells [14] for nourishment, and, thus, lactate secretion and lactate dehydrogenase (LDH; EC 1.1.1.27) activity are believed to be important parameters for assessing the functional integrity of Sertoli cells in relation to germ cell energy metabolism. Cells are normally protected against oxidative stress by multiple enzymatic mechanisms and by antioxidant molecules [15]. These biological compounds with antioxidant properties protect cells and tissues from deleterious effects of ROS and other free radicals gener-
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ating during infection [16–18]. The imbalance between free radical generation and antioxidant system can produce oxidative stress. Lipoic acid (LA) is a disulphide compound that found naturally as the coenzyme for pyruvate dehydrogenase (EC 1.2.4.1) and ␣ketoglutarate dehydrogenase (E.C. 1.2.4.2). LA and its reduced form dihydrolipoic acid are present in all kinds of microbial and eukaryotic cells and act as antioxidants not only through free radical quenching but also indirectly through recycling of other cellular antioxidants [16,19,20]. Exogenous supplementation with LA has been reported to increase unbound lipoic acid concentration. Unbound LA can act as a potent antioxidant and reduce oxidative stress both in vitro and in vivo [21]. LA is both water and fat soluble making it highly effective in reducing free radicals including lipid peroxides in cellular membranes, as well as scavenging free radicals [19]. The aim of this study was to investigate the protective effect of LA on the biochemical changes related to oxidative stress in Sertoli cells and the potential modulatory effect of LA on Sertoli cell function using the well-characterized lipopolysaccharide (LPS)-induced inflammation model [22–25]. This model mimics the human response to inflammation and infection of the male reproductive tract, leading to suppression of the hypothalamic-pituitary axis, reduction in androgen production by the Leydig cells, and disruption of the developing germ cells that correlates with the severity of the inflammation [24]. 2. Materials and methods 2.1. Animals Adult male albino rats of the ‘Wistar’ strain weighing 170 ± 10 g (90 days old) were housed in clean polypropylene cages and maintained in an air-conditioned room with constant 12 h/12 h dark and light cycle. 2.2. Reagents LPS, LA and reagents for cell culture were purchased from Sigma–Aldrich Chemical Company (St. Louis, MO, USA). Other reagents were of analytical grade. 2.3. Isolation and culture of Sertoli cells Sertoli cells were isolated as previously described [26], with some modifications. Briefly, the testes were dissociated in 1 mg/ml trypsin, 1 mg/ml collagenase (Type II, 300 U/ml) and 0.5 mg/ml hyaluronidase (Type II) in 0.01 M phosphate buffered saline (PBS) containing Ca2+ and Mg2+ by shaking (45 min, 34 ◦ C) in an orbital mixer incubator at 90 cycles/min. The tubule fragments were washed in Ca2+ - and Mg2+ -free PBS and shaken (30 min, 34 ◦ C) at 120 cycles/min. The Sertoli cell suspension was collected by centrifugation (240 g, 15 min), resuspended in PBS and filtered through a 200 m mesh. Contaminating germ cells were reduced by two successive shaken incubations (30 min, 34 ◦ C, 50 cycles/min) of the cell suspension in PBS. 2.4. Sertoli cell purity To check the purity of the culture, Sertoli cells were also grown on eight chambered glass slides. Any contaminating cell populations were analyzed before treatment with the test substance using different methods: Leydig cells were detected by staining for 3-hydroxysteroid dehydrogenase (3-HSD: [27] peritubular myoid cells were detected by histochemical staining for ␣-smooth muscle isoactin [28], and germ cells were detected by examination of cell morphology. Contamination by testicular macrophages
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was detected by the morphological appearance under the microscope [29]. Further, when Sertoli cell cultures were stained for non-specific esterase activity [30], the Sertoli cell cultures were negative and there are no microphages present. There was minor contamination by spermatogonia (3–4%), spermatocytes (2–3%), and peritubular myoid cells (1%). The viability of purified Sertoli cells was 90–95% as assessed by the trypan exclusion test [31]. 2.5. Sertoli cell culture and treatment The enriched (unbound) Sertoli cells (106 cells/ml) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham F12 ([1/1] v/v) without phenol red supplemented with gentamicin (50 g/ml), insulin (5 g/ml), and transferrin (2.5 g/ml). Cultures were placed in 6-well plates (3 ml/well) at 32 ◦ C in a humidified incubator (5% CO2 /95% air (v/v)). The medium was changed daily for 3 successive days and the cells were incubated in a transferrin-free medium containing 0.1% (w/v) bovine serum albumin (BSA) with or without the test substance. The cells were divided into 4 groups each in triplicate. Group I incubated with water (vehicle) throughout the experiment, served as a control. Group II was incubated with LA (100 M) alone for 24 h. Group III was treated with LPS (50 g/ml) alone for 12 h. Group IV was incubated (pretreatment) with LA (100 M) for 24 h then treated with LPS (50 g/ml) for 12 h. The experiment was repeated for three independent culture preparations. The dose of LPS (50 g/ml) was selected depending on the data presented by Okuma et al. [32]. At the end of incubation, Sertoli cells were recovered and sonicated in 0.1 M Tris–HCl buffer, pH 7.4 and centrifuged for 15 min at 1500 × g. The supernatant was used for all the enzymatic and non-enzymatic assays after estimating the protein content using a commercially available bicinchonic acid protein assay kit [33]. 2.6. Cell viability Cell viability was evaluated by the MTT assay [34]. At the end of treatment with LPS, 20 l of MTT (5 mg/ml in phosphate buffered saline) was added to each 96-well culture plate which was then covered with aluminum foil. After incubation for a further 4 h, the medium was removed and 100 l of DMSO was added to each well for another 2 h. The absorbance was then measured on an automated microplate reader at 570 nm. 2.7. Lactate estimation Lactate was estimated according to Krishnamoorthy et al. [12]. Briefly, 0.1 ml of cell extract was added to equal volume of 10% (w/v) TCA and the volume was completed to 1 ml with Tris–HCl buffer, pH 7.4, sonicated and centrifuged at 3000 × g for 15 min. The supernatant (1 ml) was taken in another centrifuge tube marked up to the 10 ml level. To each tube, 1 ml of 20% (w/v) copper sulphate solution was added and brought to the mark with distilled water. One gram of powdered calcium hydroxide was added; the tube was shaken vigorously and kept at room temperature for 1 h with intermittent shaking. After centrifugation, 1 ml of the supernatant or standard lactate solution (1–10 g lactate/ml) was transferred to dry tubes to which, 50 l of 4% (w/v) copper sulphate solution followed by 6 ml of concentrated sulphuric acid were added. The contents were mixed well by lateral shaking in a boiling water bath for 6.5 min and cooled. When the contents were significantly cooled, 0.1 ml of p-hydroxy diphenyl reagent was added directly into the solution and the precipitate was kept at room temperature for 30 min. Later, the contents were placed in boiling water for 30 s, followed by cooling, and the absorbance at 570 nm was compared against the reagent blank in spectrophotometer. The lactate content was calculated and expressed as mg/l × 106 cells.
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2.8. Assay of oxidative stress 2.8.1. Lipid peroxidation Malondialdehyde (MDA), formed as an end product of the oxidation of lipids, reacts with thiobarbituric acid to generate a colored product which absorbs at 532 nm. The formation of TBARS during an acid heating reaction is used as an index of intensity of oxidative stress as previously described [35]. Briefly, one volume of the test sample and two volumes of stock reagent (15% (w/v) trichloroacetic acid in 0.25N HCl and 0.37% (w/v) thiobarbituric acid in 0.25N HCl) were mixed in a centrifuge tube. The solution was heated in boiling water for 15 min. After cooling on ice, the precipitate was removed by centrifugation at 1500 × g for 10 min, and the absorbance (at 532 nm) of the supernatant was compared to a blank containing all reagents except the test sample. The malondialdehyde equivalent content of the sample was expressed as nmol/mg protein. A standard curve was constructed by extrapolating the amount of the commercially bought product malondialdehyde to the measured absorbance. The ferrous sulphate induced lipid peroxidation system contained 10 mM ferrous sulphate as inducer [36].
2.8.2. Hydrogen peroxide production Hydrogen peroxide generation was assayed by the method of Pick and Keisari [37]. Briefly, the incubation mixture contained 1.641 ml of phosphate buffer (50 mM, pH 7.6), 54 l of horse radish peroxidase (8.5 units/ml), 30 l of phenol red (0.28 nM), 165 l of dextrose (5.5 nM) and 100 l of cell extract. The reaction was incubated at 35 ◦ C for 30 min and was terminated by the addition of 60 l of 10N sodium hydroxide. The absorbance was read at 610 nm. The quantity of H2 O2 produced was expressed as mol of H2 O2 generated/min/mg protein. For the standard curve, a known amount of hydrogen peroxide and all the above reagents except the enzyme extract were incubated for 30 min at 35 ◦ C, terminated by the addition of 60 l of 10N sodium hydroxide and the absorbance read at 610 nm.
2.9. Antioxidant enzymes assay 2.9.1. Superoxide dismutase (SOD) Superoxide dismutase activity was assessed as described [38]. Briefly, the assay mixture contained 1 ml of 0.1 mol/l Tris–HCl buffer (pH 8.2), 0.25 ml of 2 mmol/l pyrogallol, 50 l of the enzyme extract and water to give a final volume of 2 ml. The rate of inhibition of pyrogallol auto-oxidation after the addition of enzyme extract was noted. The enzyme activity was expressed as U/mg protein, with one unit of the enzyme activity defined as the amount required for 50% inhibition of pyrogallol auto-oxidation.
2.9.2. Catalase Catalase activity was assayed by the method of Sinha [39]. Briefly, the assay mixture containing 0.5 ml of 0.2 M H2 O2 , 1 ml of sodium phosphate buffer (pH 7.0, 0.01 M) and 0.4 ml of water. After that 0.1 ml of cell extract was added to initiate the reaction. Then 2 ml of dichromate-acetic acid reagent was added after 15, 30, 45 and 60 s, to arrest the reaction. To the control tube the enzyme was added after the addition of dichromate-acetic acid reagent. The tubes were then heated for 10 min, allowed to cool, and the green color that developed was read at 570 nm against a blank, containing all components except the enzyme, in a spectrophotometer. The activity of catalase was expressed as mol of H2 O2 consumed/min/mg protein (1 U is the amount of enzyme that utilizes mol of hydrogen peroxide/min).
2.9.3. Glutathione peroxidase (GSH-Px; GPx) Glutathione peroxidase activity was determined as described [40] based on the reaction between glutathione remaining after the action of GPx and 5,5 -dithiobis-(2-nitrobenzoic acid) to give a complex that absorbs at 412 nm. The enzyme activity was expressed as U/mg protein (1 U is the amount of enzyme that converts mol GSH to GSSG in the presence of hydrogen peroxide/min). 2.9.4. Glutathione reductase (GR) Glutathione reductase uses NADPH to convert oxidized glutathione (GSSG) to the reduced form (GSH). Its activity was determined by the method of Staal et al. [41]. Briefly, the assay mixture containing 0.2 ml of cell extract, 1.5 ml of sodium phosphate buffer, pH 7.0, 0.5 ml of 25 mM EDTA, 0.2 ml of 12.5 mM oxidized glutathione, and 0.1 ml of 3 mM NADPH were added and the decrease in absorbance at 340 nm compared for 1 min against blank containing all the components except cell extract in a spectrophotometer was measured. Activity was expressed as nmol of NADPH oxidized/min/mg protein. One unit of GR activity was defined as the amount of the enzyme that catalyzes the oxidation of mol of NADPH per minute. 2.9.5. Glutathione-S-transferase (GST) The activity of GST was assayed by the method of Habig et al. [42]. To 0.4 ml potassium phosphate buffer (0.5 mol/l; pH 6.5), 50 l of cell extract, 1.2 ml water and 0.1 ml CDNB (1-chloro-2,4dinitrobenzene, 30 mmol/l) conjugate were added and incubated in a water bath at 37 ◦ C for 10 min. After incubation, 0.1 ml of reduced glutathione (30 mmol/l) was added. The change in optical density was measured against blank at 340 nm at 30 s interval. Activity of GST was expressed as U/mg protein (1 U is the amount of enzyme that conjugates mol of 1-chloro-2,4-dinitrobenzene with GSH/min). 2.10. Assay of Sertoli cell enzymes 2.10.1. -GT Activity was determined by using l-␥-glutamyl-p-nitroanilide as substrate [43]. The colorless substrate exhibits maximum absorbance at 315 nm, while free p-nitroaniline is yellow and exhibits high absorbance between 350 and 420 nm. Enzymatic release of p-nitroaniline was followed at 410 nm, at which wavelength the substrate exhibits no absorbance. Determination of enzymatic activity was performed by adding 0.1 ml of cell extract to 0.9 ml of a solution (at 37 ◦ C) containing l-␥-glutamyl-pnitroanilide (5 mol), magnesium chloride (10 mol) and Tris–HCl buffer (pH 9.0; 100 mol). After 3–5 min of incubation, the reaction was terminated by addition of 2 ml of 1.5N acetic acid, and the absorbance was measured at 410 nm against a reference solution containing the same components except that the enzyme was added after addition of acetic acid. Enzymatic activity was determined by direct spectrophotometric measurement. The rate of generation of p-nitroaniline was found to be linear over that time. The activity of the enzyme was expressed as mol of p-nitroaniline formed during 1 min of incubation per mg of protein (1 U of enzyme activity was defined as mol of p-nitroaniline formed per minute per milligram of protein (mol/min/mg protein). 2.10.2. ˇ-Glucuronidase -Glucuronidase activity was determined by measuring the absorbance at 540 nm of phenolphthalein released from the substrate phenolphthalein glucuronide by the spectrophotometric method of Fishmann [44]. Total reaction volume was 150 l. The reaction mixture contained 50 l of reagent A (75 mM potassium phosphate buffer, with 1.0% (w/v) bovine serum albumin, pH 6.8 at 37 ◦ C), 25 l of reagent B (3.0 mM phenolphthalein glucuronide
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Table 1 Effect of LPS and lipoic acid on Sertoli cells viability.
Cell viability (%)
Group I control
Group II (LA)
Group III (LPS)
Group IV (LA + LPS)
91.33 ± 1.53
94.33 ± 1.15
90.67 ± 2.08
93.67 ± 0.58
Values are expressed as mean ± SD (n = 3 in each group). Comparisons are made between; Group I and Groups II–IV.
substrate solution), mix by inversion and equilibrate to 37 ◦ C. Then 10 l of cell extract were added and incubate at 37 ◦ C for exactly 30 min. The reaction was stopped by adding 500 l of 200 mM glycine buffer solution, pH 10.4. One unit of activity was defined as mol of phenolphthalein liberated per minute per milligram of protein (mol/min/mg protein). 2.10.3. Non-enzymatic antioxidant assay Total reduced glutathione (GSH) was estimated by the method of Sedalk and Lindsay [45], where the absorbance was measured at 412 nm. The amount of GSH was determined from a standard curve, under the same conditions, with a standard solution of GSH. GSH concentration was expressed as nmol/mg protein. Ascorbic acid (vitamin C) was assayed by the method of Omaye et al. [46]. Vitamin C was oxidized by copper to form dehydroascorbic acid which reacts with 2,4-dinitrophenylhydrazine to form the derivative bis2,4-dinitrophenylhydrazone. 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. The concentration of vitamin C was expressed as g/mg protein.
Fig. 1. Effect of lipopolysaccharide and lipoic acid on hydrogen peroxide production in adult rat Sertoli cells. Group I incubated with water (vehicle) throughout the experiment, served as a control. Group II was incubated with LA (100 M) alone for 24 h. Group III was treated with LPS (50 g/ml) alone for 12 h. Group IV was incubated (pretreatment) with (100 M) for 24 h then treated with LPS (50 g/ml) for 12 h. Values are mean ± SD (n = 3). Statistical analysis (ANOVA) for differences from corresponding control: comparisons were made between: a Group I and Groups II–IV; b Group III and Group IV. The symbols represent statistical significance from control where * P < 0.05; *** was a P < 0.001.
3.2. Lipid peroxidation (LPO) and hydrogen peroxide (H2 O2 ) production
2.11. Statistical analysis Results were expressed as mean ± standard deviation (SD), n = 3 (three independent culture preparations, each in triplicate). Differences in mean were analyzed by one-way analysis of variance (ANOVA). If F-value was significant, the data were subjected to Tukey–Kramer multiple comparison test. P < 0.05 was considered statistically significant. 3. Results
LPS-treated cells (Group III) showed increase in the amount of H2 O2 (96.5%), but not after LA pretreatment (Group IV; Fig. 1). There was also an abnormal increase of LPO in the LPS-treated Sertoli cells. The Group III cells displayed a 69% rise in basal LPO in cellular protein, as well as 53% in LPO following addition of the exogenous inducer ferrous sulphate compared to the control Group 1 (Table 1). LA pretreatment (Group IV) suppressed the increase in LPO induced by LPS (P < 0.05) (Table 2).
This study reported that at doses up to 500 g/ml for 24 h, LPS stimulated activin A and IL-1␣ (pro-inflammatory cytokine) secretion and inhibited inhibin B secretion by adult Sertoli cell cultures, and a maximum stimulatory effect was observed at a dose below 25 g/ml. Moreover, this study presented that higher doses of LPS (>500 g/ml) were toxic to the Sertoli cells, as indicated by the loss of cellular processes, rounding and detachment of the cells in culture.
3.3. Non-enzymatic antioxidants
3.1. Cell viability
3.4. Non-enzymatic antioxidants
The results showed that LPS did not significantly affect Sertoli cell viability in any of the experimental groups under our experimental conditions (Table 1).
Changes in Sertoli cell non-enzymatic antioxidants were shown in Table 3. LPS-treated cells (Group III) demonstrated a decrease in the concentration of GSH and antioxidant vitamin C (P < 0.001). LA
3.3.1. Lactate production The Sertoli cellular lactate concentration was significantly decreased (43%) in LPS-treated cells (Group III) when compared to control; however, supplementation of lipoic acid showed a significant increase in lactate concentration when compared to LPS-treated cells (P < 0.001) (Fig. 2).
Table 2 Effect of LPS and lipoic acid on lipid peroxidation in adult rat Sertoli cells. Lipid peroxidation
Group I control
Group II (LA)
Group III (LPS)
Group IV (LA + LPS)
Basal FeSO4 -induced
2.33 ± 0.21 7.6 ± 0.44
2.23 ± 0.21 7.9 ± 0.17
3.93 ± 0.15a , *** 11.6 ± 0.36a , ***
2.83 ± 0.15a , * , *** 8.1 ± 0.2b , ***
Values are expressed as mean ± SD (n = 3 in each group). Units—LPO: nM of MDA equivalent formed/mg protein. a Comparisons are made between; Group I and Groups II–IV. b Comparisons are made between; Group VIII and Group IV. * The symbols represent statistical significance from control: P < 0.05. *** The symbols represent statistical significance from control: P < 0.001.
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Fig. 2. Effect of lipopolysaccharide and lipoic acid on lactate concentration in adult rat Sertoli cells. Group I incubated with water (vehicle) throughout the experiment, served as a control. Group II was incubated with LA (100 M) alone for 24 h. Group III was treated with LPS (50 g/ml) alone for 12 h. Group IV was incubated (pretreatment) with (100 M) for 24 h then treated with LPS (50 g/ml) for 12 h. Values are mean ± SD (n = 3). Statistical analysis (ANOVA) for differences from corresponding control: comparisons were made between: a Group I and Groups II–IV; b Group III and Group IV. The symbols represent statistical significance from control where *** was a P < 0.001.
pretreatment maintained GSH and vitamin C concentrations near normal amounts. 3.5. Enzymatic antioxidants
Fig. 3. Effect of lipopolysaccharide and lipoic acid on ␥-GT (A) and -glucuronidase activity (B) in adult rat Sertoli cells. Group I incubated with water (vehicle) throughout the experiment, served as a control. Group II was incubated with LA (100 M) alone for 24 h. Group III was treated with LPS (50 g/ml) alone for 12 h. Group IV was incubated (pretreatment) with (100 M) for 24 h then treated with LPS (50 g/ml) for 12 h. Values are mean ± SD (n = 3). Statistical analysis (ANOVA) for differences from corresponding control: comparisons were made between: a Group I and Groups II–IV; b Group III and Group IV. The symbols represent statistical significance from control where *** was a P < 0.001.
Table 4 showed the activities of the antioxidant enzymes in Sertoli cell homogenate. LPS-treatment (Group III) decreased the specific activities of SOD (66%), CAT (39%), GPX (49%), GR (51%) and GST (42%) in comparison to control activities (Group I). All the LPSinduced alterations in the antioxidant enzymatic activities were effectively prevented by LA pretreatment. Moreover, LA-treated cells showed enhanced antioxidant capacity (Group II).
4. Discussion 3.6. Sertoli cell enzymes Bacterial infection of the genital tract may originate from the urinary tract or can be sexually transmitted [47,48]. This pathological infection will create an acute inflammatory response with an influx of leukocytes into the genital tract and a resulting increase in oxygen and oxygen-derived oxidant (ROS) production which may lead to oxidative stress [49–51]. These ROS include oxygen
The activities of ␥-GT and -glucuronidase were significantly decreased in LPS-treated cells (Group III) by 48% and 35% respectively compared to control (Group I). The activity of these enzymes was normalized by comparison to the LA pretreatment (Group IV, Fig. 3). Table 3 Effect of LPS and lipoic acid on non-enzymic antioxidants in adult rat Sertoli cells. Non-enzymic antioxidant
Group I control
Group II (LA)
Group III (LPS)
Group IV (LA + LPS)
GSH (nmol/mg protein) Vitamin C (g/mg protein)
11.47 ± 0.25 1.2 ± 0.1
12.5 ± 0.4 * 1.22 ± 0.01
5.57 ± 0.35 0.61 ± 0.03a , ***
11.03 ± 0.32b , *** 1.03 ± 0.06a , * , b , ***
a,
a , ***
Values represent mean ± SD (n = 3 in each group). a Comparisons are made between; Group I and Groups II–IV. b Comparisons are made between; Group VIII and Group IV. * The symbols represent statistical significance from control: P < 0.05. *** The symbols represent statistical significance from control: P < 0.001.
Table 4 Effect of LPS and lipoic acid on the activities of Sertoli cells antioxidant enzymes. Enzymatic antioxidants
Group I control
SOD CAT GPX GR GST
7 39.67 73 0.45 0.045
± ± ± ± ±
0.2 1.53 3 0.03 0.001
Group II (LA) 7.3 40.2 76.33 0.47 0.046
± ± ± ± ±
0.3 2.43 2.08 0.03 0.002
Group III (LPS) 2.37 24.3 37.33 0.22 0.026
± ± ± ± ±
0.15a , *** 1.4a , *** 2.52a , *** 0.03a , *** 0.003a , ***
Group IV (LA + LPS) 6.83 37.6 70.77 0.43 0.044
± ± ± ± ±
0.45b , *** 1.45b , *** 1.06b , *** 0.01b , *** 0.002b , ***
Values are expressed as mean ± SD (n = 3 in each group). Units—SOD: units/mg protein (1 U = amount of enzyme that inhibits the auto-oxidation of pyrogallol by 50%); CAT:.M of H2 O2 consumed/min/mg protein; GPX: M of reduced glutathione consumed/min/mg protein; GR: M of NADPH oxidized/min/mg protein; GST: M of 1-chloro-2,4dinitrobenzene-GSH conjugate formed/min/mg protein; ␥-GT: M of p-nitroaniline formed/min/mg protein. a Comparisons were made between: Group I and Groups II–IV. b Comparisons were made between: Group III and Group IV. *** The symbols represent statistical significance from control: P < 0.001.
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free radicals such as superoxide, hydroxyl, peroxyl, alkoxyl and hydroperoxyl radicals which cause tissue and cellular damage by a variety of mechanisms including DNA damage, lipid peroxidation, protein peroxidation, depletion of antioxidant enzymes and non-enzymatic antioxidant molecules. Evidence has implicated oxidative stress as an etiological factor in the development of male infertility [52]. In addition, Amin [53] reported that ROS are involved in a variety of patho-physiological conditions of testes. Sertoli cells create a microenvironment that protects seminiferous tubules from autoantigens and invading pathogens [54]. Previous studies have demonstrated that Sertoli cells produce the pro-inflammatory cytokines IL-1␣ and IL-6 and their secretion is stimulated by LPS, through stimulation of Toll-like receptors (TLRs) which play crucial roles in mediating innate and adaptive immunity [54]. Here, the protective effects of LA were shown. ␣-Lipoic acid is a racemic mixture R(+)-␣-LA is a cofactor of the pyruvate dehydrogenase and ␣-dehydrogenase complexes. Moreover, enantiomers of LA and their reduced form (dihydrolipoic acid) act as extraand intracellular redox couples and powerful lipophilic free radical scavengers. LA is highly reactive with free radicals and capable of increasing concentrations of GSH [55]. It has been shown that LA reduces oxidative stress in healthy adults and diabetic patients by decreasing significantly lipid peroxidation [55]. Therefore, the mechanism of LA protection for Sertoli cells can be inferred. In the present study, the decrease in Sertoli cellular lactate in LPS-treated cells may be due to oxygen toxicity and cytotoxicity through hydrogen peroxide that alters normal cellular metabolic function leading to a decrease in glycolysis. Several studies have indicated that lactate represents a preferential energy substrate for germ cells [56–58]. Lactate is produced in Sertoli cells from pyruvate following lactate dehydrogenase A (LDHA) action in Sertoli cells and is transported across the plasma membrane to the germ cells by specific proton/monocarboxylate transporters (MCTs) [59]. Lactate is then converted into pyruvate in germ cells following lactate dehydrogenase C (LDHC) action [60]. LPS-induced inflammation had a marked oxidative impact as evidenced by the significant increase of LPO and H2 O2 concentration. This change might have resulted from increased production of ROS and/or a decrease in antioxidant status. The high concentrations of polyunsaturated fatty acids in Sertoli cells caused susceptibility to lipid peroxidative degradation. The increased LPO due to LPS treatments was a consequence of impaired antioxidant enzyme activities and depletion in GSH and vitamin C concentrations. LPS-treated Sertoli cells may be more susceptible to LPO in the presence of promoter like ferrous sulphate. The elevated amounts of lipid peroxides that resulted from increased free radical formation might provide a biochemical mechanism for inflammation-associated testicular side effects. The prooxidant-antioxidant balance in LPS-induced inflammation was shifted toward prooxidants; hence, exogenous administration of a potential antioxidant such as LA was expected to normalize the prooxidant-antioxidant status. The ability of LA or its reduced form to scavenge free radicals may have restricted the production of toxic lipid peroxides and so reduced cellular need for enzymatic antioxidants. ROS enzymatic scavengers like SOD, CAT, GPx, GST and GR and antioxidants like GSH and vitamin C may protect cells from the injurious effects of ROS [61,15]. The decrease in activities of enzymatic antioxidants in LPS-induced inflammation in Sertoli cells might have increased LPO and ROS production. A decrease in SOD activity has been shown to increase the concentration of superoxide anions, which is known to inactivate GPx [62]. Equally, when GPx fails to eliminate H2 O2 from the cell the accumulated H2 O2 has been shown to cause inactivation of SOD [62]. Thus the balance of enzymes and pathways may be essential defenses against the superoxide anion and peroxides generated in
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the Sertoli cells. GST is inactivated either due to excessive production of H2 O2 [63] and/or reduced GSH amounts [63] as observed here. Thiols are the major components of cellular antioxidant system that play an important role as antioxidant against ROS and free radicals. GSH and its oxidized form glutathione disulphide constitute a major redox buffer system of the cell [64]. GSH is the major intracellular non-protein sulphydryl and its depletion increased the susceptibility of cells to oxidative stress [65]. Further, the decreased GSH content may explain the decreased concentration of vitamin C, which enters the cells mainly in the oxidized form where it is reduced by GSH [66]. The decrease in the concentration of this vitamin may have serious consequences as, in addition to antioxidant function, it plays a role in regenerating other antioxidants [66]. In summary, here LPS-induced inflammation in Sertoli cells induced significant oxidative stress that was associated with decreased enzymatic and non-enzymatic antioxidant molecules and the prooxidant-antioxidant balance was shifted towards prooxidants. Lipoic acid supplementation reduced oxidative stress in Sertoli cells treated with LPS by alleviating LPO through scavenging of free radicals or by enhancing the activities of antioxidant enzymes as well as the concentrations of vitamin C and GSH which then detoxify free radicals. Conflict of interest No conflicts of interest. References [1] M.E. Cutolo, M.K. Chan, H.Z. Movat, Sex hormone status of patients with rheumatoid arthritis: evidence of low serum concentrations at baseline and after human chorionic gonadotrophin stimulation, Arthritis Rheum. 31 (1988) 1314–1317. [2] J.P. Buch, S.K. Havlovec, Variation in sperm penetration assay related to viral illness, Fertil. Steril. 55 (1999) 844–846. [3] M. Shekarriz, A.J. Thomas, A. Agarwal, Incidence and level of seminal reactive oxygen species in normal men, Urology 45 (1995) 103–107. [4] A. Agarwal, S. Prabakaran, S. Allamanen, What an andrologist/urologist should know about free radicals and why, Urology 67 (2006) 2–8. [5] A. Agarwal, K. Makker, R. Sharma, Clinical relevance of oxidative stress in male factor infertility: an update, Am. J. Reprod. Immunol. 59 (2008) 2–11. [6] J.P. Buch, D.J. Lamb, L.I. Lipshultz, R.G. Smith, Partial characterization of a unique growth factor secreted by human Sertoli cells, Fertil. Steril. 49 (1988) 658–665. [7] A.R. Bellve, W. Zheng, Growth factors as autocrine and paracrine modulators of male gonadal functions, J. Reprod. Fertil. 85 (1989) 771–793. [8] M. Dym, D.W. Fawcett, The blood–testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium, Biol. Reprod. 3 (1970) 308–326. [9] W.Y. Lui, D. Mruk, W.M. Lee, C.Y. Cheng, Sertoli cell tight junction dynamics: their regulation during spermatogenesis, Biol. Reprod. 68 (2003) 1087–1097. [10] B.P. Setchell, The testis and tissue transplantation: historical aspects, J. Reprod. Immunol. 18 (1990) 1–8. [11] A. Steinberger, G. Klinefelter, Sensitivity of Sertoli and Leydig cells to xenobiotics in in vitro models, Reprod. Toxicol. 7 (1993) 23–37. [12] G. Krishnamoorthy, P. Murugesan, R. Muthuvel, D.N. Gunadharini, A.R. Vijayababu, A. Arunkumar, P. Venkataraman, M.M. Aruldhas, J. Arunakaran, Effect of Aroclor 1254 on Sertoli cellular antioxidant system, androgen binding protein and lactate in adult rat in vitro, Toxicology 212 (2005) 195–205. [13] S. Srivastava, P.K. Seth, S.P. Srivastava, Effect of styrene administration on rat testis, Arch. Toxicol. 63 (1989) 43–46. [14] A.J. Grootegoed, P. Jansen, H.J. Vander Molen, The role of glucose, pyruvate and lactate in ATP production by rat spermatocyte and spermatid, Biochem. Biophys. Acta 762 (1984) 248–256. [15] F. Bauché, M.H. Fouchard, B. Jégou, Antioxidant system in rat testicular cells, FEBS Lett. 349 (1994) 392–396. [16] L. Packer, E.H. Witt, H.J. Tritschler, Alpha-lipoic acid as a biological antioxidant, Free Radic. Biol. Med. 19 (1995) 227–250. [17] K. Jacob, M.J. Periago, V. Böhm, G.R. Berruezo, Influence of lycopene and vitamin C from tomato juice on biomarkers of oxidative stress and inflammation, Br. J. Nutr. 99 (2008) 137–146. [18] S. Abe, Y. Tanaka, N. Fujise, T. Nakamura, H. Masunaga, T. Nagasawa, M. Yagi, An antioxidative nutrient-rich enteral diet attenuates lethal activity and oxidative stress induced by lipopolysaccharide in mice, JPEN J. Parenter. Enteral Nutr. 31 (2007) 181–187. [19] G.P. Biewenga, G.R. Haenen, A. Bast, The pharmacology of the antioxidant lipoic acid, Gen. Pharmacol. 29 (1997) 315–331.
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Modulatory role of lipoic acid on lipopolysaccharide-induced oxidative stress in adult rat Sertoli cells in vitro Hamdy A.A. Aly a,b , David A. Lightfoot c , Hany A. El-Shemy d,∗ a
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt Unit of Cellular and Molecular Pharmacology, Catholic University of Louvain, B-1200 Brussels, Belgium Genomics Core Facility, Department of Plant Soil and Agricultural Systems, SIUC, Carbondale, IL, USA d Faculty of Agriculture Research Park (FARP) and Department of Biochemistry, Faculty of Agriculture, Cairo University, 12513 Giza, Egypt b c
a r t i c l e
i n f o
Article history: Received 25 June 2009 Received in revised form 14 August 2009 Accepted 17 August 2009 Available online 21 August 2009 Keywords: Lipoic acid Sertoli cells Inflammation LPS Testicular toxicity Lipid peroxidation Antioxidants
a b s t r a c t Inflammatory reactions to microbial infections may cause male infertility. The mechanisms of inhibition of spermatogenesis can be studied in vitro using rat Sertoli cells. Bacterial lipopolysaccharides (LPS) induce acute inflammations. So LPS treated Sertoli cells can be used to test for new therapeutic compounds. The present study aimed to investigate the protective efficacy of dl-␣-lipoic acid (LA) on lipopolysaccharide (LPS)-induced oxidative stress in adult rat Sertoli cells. Sertoli cells were divided into 4 groups. Group I served as a control incubated with water (vehicle). Groups II and IV were incubated with 100 M LA for 24 h before incubating Groups III and IV with 50 g/ml lipopolysaccharide (LPS) for 12 h. In Group III cells (LPS-treated, no LA) the lactate concentration was decreased whereas hydrogen peroxide production and lipid peroxidation were significantly increased. Moreover, the activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, catalase, glutathioneS-transferase, glutathione reductase were reduced. The concentrations of antioxidant molecules such as reduced glutathione and vitamin C were significantly decreased. The activities of enzymes normally elevated in Sertoli cells, ␥-glutamyl transpeptidase and -glucuronidase, were significantly decreased. Treatment with LA (100 M) for 24 h before LPS-treatment (Group IV), prevented these changes in enzyme activities and metabolite concentrations. Therefore, LA may have a cyto-protective role during LPS-induced inflammation in adult rat Sertoli cells. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction There is a considerable body of clinical evidence suggesting that testis function is decreased during illness or infection, resulting in a temporary or permanent impairment of fertility [1,2]. This impairment is manifested as a decrease in both serum testosterone concentration and sperm counts. The precise mechanism underlying this inhibition is not clearly understood. Of the many causes of male infertility, oxidative stress (OS), a condition mediated by reactive oxygen species (ROS), has been attributed to affect the fertility status. Consequently, OS has been studied extensively in recent years. Evidence now suggests that ROS is a significant contributing pathology in 30–80% of male infertility cases [3–5]. The Sertoli cells play a major role in regulation of spermatogenesis and altering rates of spermatozoa produced. Functions include; providing structural support and nutrition to developing germ cells; phagocytosis of degenerating germ cells and residual bodies; release of spermatids at spermiation; and production of a
∗ Corresponding author. E-mail address: [email protected] (H.A. El-Shemy). 0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2009.08.013
host of proteins that regulate and/or respond to pituitary hormone release; and influence mitotic activity of spermatogonia [6,7]. Tight junctions between adjacent Sertoli cells create the blood–testis barrier, which is the barrier between germ cells situated within the basal and adluminal compartments [8,9]. Protection of developing germ cells, mediated by Sertoli cells, from harmful influences has a high priority in all species [10]. Sertoli cells have long been known to be the targets for various toxicants and infectious agents [11] and a well-established model for toxicity testing in male reproductive systems, both in vivo and in vitro [12]. -Glucuronidase (EC 3.2.1.31) and ␥-glutamyl transpeptidase (␥-GT; 2.3.2.2) activities are considered as important markers of Sertoli cell function [13]. Mature germ cells depend critically on a supply of lactate by Sertoli cells [14] for nourishment, and, thus, lactate secretion and lactate dehydrogenase (LDH; EC 1.1.1.27) activity are believed to be important parameters for assessing the functional integrity of Sertoli cells in relation to germ cell energy metabolism. Cells are normally protected against oxidative stress by multiple enzymatic mechanisms and by antioxidant molecules [15]. These biological compounds with antioxidant properties protect cells and tissues from deleterious effects of ROS and other free radicals gener-
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ating during infection [16–18]. The imbalance between free radical generation and antioxidant system can produce oxidative stress. Lipoic acid (LA) is a disulphide compound that found naturally as the coenzyme for pyruvate dehydrogenase (EC 1.2.4.1) and ␣ketoglutarate dehydrogenase (E.C. 1.2.4.2). LA and its reduced form dihydrolipoic acid are present in all kinds of microbial and eukaryotic cells and act as antioxidants not only through free radical quenching but also indirectly through recycling of other cellular antioxidants [16,19,20]. Exogenous supplementation with LA has been reported to increase unbound lipoic acid concentration. Unbound LA can act as a potent antioxidant and reduce oxidative stress both in vitro and in vivo [21]. LA is both water and fat soluble making it highly effective in reducing free radicals including lipid peroxides in cellular membranes, as well as scavenging free radicals [19]. The aim of this study was to investigate the protective effect of LA on the biochemical changes related to oxidative stress in Sertoli cells and the potential modulatory effect of LA on Sertoli cell function using the well-characterized lipopolysaccharide (LPS)-induced inflammation model [22–25]. This model mimics the human response to inflammation and infection of the male reproductive tract, leading to suppression of the hypothalamic-pituitary axis, reduction in androgen production by the Leydig cells, and disruption of the developing germ cells that correlates with the severity of the inflammation [24]. 2. Materials and methods 2.1. Animals Adult male albino rats of the ‘Wistar’ strain weighing 170 ± 10 g (90 days old) were housed in clean polypropylene cages and maintained in an air-conditioned room with constant 12 h/12 h dark and light cycle. 2.2. Reagents LPS, LA and reagents for cell culture were purchased from Sigma–Aldrich Chemical Company (St. Louis, MO, USA). Other reagents were of analytical grade. 2.3. Isolation and culture of Sertoli cells Sertoli cells were isolated as previously described [26], with some modifications. Briefly, the testes were dissociated in 1 mg/ml trypsin, 1 mg/ml collagenase (Type II, 300 U/ml) and 0.5 mg/ml hyaluronidase (Type II) in 0.01 M phosphate buffered saline (PBS) containing Ca2+ and Mg2+ by shaking (45 min, 34 ◦ C) in an orbital mixer incubator at 90 cycles/min. The tubule fragments were washed in Ca2+ - and Mg2+ -free PBS and shaken (30 min, 34 ◦ C) at 120 cycles/min. The Sertoli cell suspension was collected by centrifugation (240 g, 15 min), resuspended in PBS and filtered through a 200 m mesh. Contaminating germ cells were reduced by two successive shaken incubations (30 min, 34 ◦ C, 50 cycles/min) of the cell suspension in PBS. 2.4. Sertoli cell purity To check the purity of the culture, Sertoli cells were also grown on eight chambered glass slides. Any contaminating cell populations were analyzed before treatment with the test substance using different methods: Leydig cells were detected by staining for 3-hydroxysteroid dehydrogenase (3-HSD: [27] peritubular myoid cells were detected by histochemical staining for ␣-smooth muscle isoactin [28], and germ cells were detected by examination of cell morphology. Contamination by testicular macrophages
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was detected by the morphological appearance under the microscope [29]. Further, when Sertoli cell cultures were stained for non-specific esterase activity [30], the Sertoli cell cultures were negative and there are no microphages present. There was minor contamination by spermatogonia (3–4%), spermatocytes (2–3%), and peritubular myoid cells (1%). The viability of purified Sertoli cells was 90–95% as assessed by the trypan exclusion test [31]. 2.5. Sertoli cell culture and treatment The enriched (unbound) Sertoli cells (106 cells/ml) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham F12 ([1/1] v/v) without phenol red supplemented with gentamicin (50 g/ml), insulin (5 g/ml), and transferrin (2.5 g/ml). Cultures were placed in 6-well plates (3 ml/well) at 32 ◦ C in a humidified incubator (5% CO2 /95% air (v/v)). The medium was changed daily for 3 successive days and the cells were incubated in a transferrin-free medium containing 0.1% (w/v) bovine serum albumin (BSA) with or without the test substance. The cells were divided into 4 groups each in triplicate. Group I incubated with water (vehicle) throughout the experiment, served as a control. Group II was incubated with LA (100 M) alone for 24 h. Group III was treated with LPS (50 g/ml) alone for 12 h. Group IV was incubated (pretreatment) with LA (100 M) for 24 h then treated with LPS (50 g/ml) for 12 h. The experiment was repeated for three independent culture preparations. The dose of LPS (50 g/ml) was selected depending on the data presented by Okuma et al. [32]. At the end of incubation, Sertoli cells were recovered and sonicated in 0.1 M Tris–HCl buffer, pH 7.4 and centrifuged for 15 min at 1500 × g. The supernatant was used for all the enzymatic and non-enzymatic assays after estimating the protein content using a commercially available bicinchonic acid protein assay kit [33]. 2.6. Cell viability Cell viability was evaluated by the MTT assay [34]. At the end of treatment with LPS, 20 l of MTT (5 mg/ml in phosphate buffered saline) was added to each 96-well culture plate which was then covered with aluminum foil. After incubation for a further 4 h, the medium was removed and 100 l of DMSO was added to each well for another 2 h. The absorbance was then measured on an automated microplate reader at 570 nm. 2.7. Lactate estimation Lactate was estimated according to Krishnamoorthy et al. [12]. Briefly, 0.1 ml of cell extract was added to equal volume of 10% (w/v) TCA and the volume was completed to 1 ml with Tris–HCl buffer, pH 7.4, sonicated and centrifuged at 3000 × g for 15 min. The supernatant (1 ml) was taken in another centrifuge tube marked up to the 10 ml level. To each tube, 1 ml of 20% (w/v) copper sulphate solution was added and brought to the mark with distilled water. One gram of powdered calcium hydroxide was added; the tube was shaken vigorously and kept at room temperature for 1 h with intermittent shaking. After centrifugation, 1 ml of the supernatant or standard lactate solution (1–10 g lactate/ml) was transferred to dry tubes to which, 50 l of 4% (w/v) copper sulphate solution followed by 6 ml of concentrated sulphuric acid were added. The contents were mixed well by lateral shaking in a boiling water bath for 6.5 min and cooled. When the contents were significantly cooled, 0.1 ml of p-hydroxy diphenyl reagent was added directly into the solution and the precipitate was kept at room temperature for 30 min. Later, the contents were placed in boiling water for 30 s, followed by cooling, and the absorbance at 570 nm was compared against the reagent blank in spectrophotometer. The lactate content was calculated and expressed as mg/l × 106 cells.
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2.8. Assay of oxidative stress 2.8.1. Lipid peroxidation Malondialdehyde (MDA), formed as an end product of the oxidation of lipids, reacts with thiobarbituric acid to generate a colored product which absorbs at 532 nm. The formation of TBARS during an acid heating reaction is used as an index of intensity of oxidative stress as previously described [35]. Briefly, one volume of the test sample and two volumes of stock reagent (15% (w/v) trichloroacetic acid in 0.25N HCl and 0.37% (w/v) thiobarbituric acid in 0.25N HCl) were mixed in a centrifuge tube. The solution was heated in boiling water for 15 min. After cooling on ice, the precipitate was removed by centrifugation at 1500 × g for 10 min, and the absorbance (at 532 nm) of the supernatant was compared to a blank containing all reagents except the test sample. The malondialdehyde equivalent content of the sample was expressed as nmol/mg protein. A standard curve was constructed by extrapolating the amount of the commercially bought product malondialdehyde to the measured absorbance. The ferrous sulphate induced lipid peroxidation system contained 10 mM ferrous sulphate as inducer [36].
2.8.2. Hydrogen peroxide production Hydrogen peroxide generation was assayed by the method of Pick and Keisari [37]. Briefly, the incubation mixture contained 1.641 ml of phosphate buffer (50 mM, pH 7.6), 54 l of horse radish peroxidase (8.5 units/ml), 30 l of phenol red (0.28 nM), 165 l of dextrose (5.5 nM) and 100 l of cell extract. The reaction was incubated at 35 ◦ C for 30 min and was terminated by the addition of 60 l of 10N sodium hydroxide. The absorbance was read at 610 nm. The quantity of H2 O2 produced was expressed as mol of H2 O2 generated/min/mg protein. For the standard curve, a known amount of hydrogen peroxide and all the above reagents except the enzyme extract were incubated for 30 min at 35 ◦ C, terminated by the addition of 60 l of 10N sodium hydroxide and the absorbance read at 610 nm.
2.9. Antioxidant enzymes assay 2.9.1. Superoxide dismutase (SOD) Superoxide dismutase activity was assessed as described [38]. Briefly, the assay mixture contained 1 ml of 0.1 mol/l Tris–HCl buffer (pH 8.2), 0.25 ml of 2 mmol/l pyrogallol, 50 l of the enzyme extract and water to give a final volume of 2 ml. The rate of inhibition of pyrogallol auto-oxidation after the addition of enzyme extract was noted. The enzyme activity was expressed as U/mg protein, with one unit of the enzyme activity defined as the amount required for 50% inhibition of pyrogallol auto-oxidation.
2.9.2. Catalase Catalase activity was assayed by the method of Sinha [39]. Briefly, the assay mixture containing 0.5 ml of 0.2 M H2 O2 , 1 ml of sodium phosphate buffer (pH 7.0, 0.01 M) and 0.4 ml of water. After that 0.1 ml of cell extract was added to initiate the reaction. Then 2 ml of dichromate-acetic acid reagent was added after 15, 30, 45 and 60 s, to arrest the reaction. To the control tube the enzyme was added after the addition of dichromate-acetic acid reagent. The tubes were then heated for 10 min, allowed to cool, and the green color that developed was read at 570 nm against a blank, containing all components except the enzyme, in a spectrophotometer. The activity of catalase was expressed as mol of H2 O2 consumed/min/mg protein (1 U is the amount of enzyme that utilizes mol of hydrogen peroxide/min).
2.9.3. Glutathione peroxidase (GSH-Px; GPx) Glutathione peroxidase activity was determined as described [40] based on the reaction between glutathione remaining after the action of GPx and 5,5 -dithiobis-(2-nitrobenzoic acid) to give a complex that absorbs at 412 nm. The enzyme activity was expressed as U/mg protein (1 U is the amount of enzyme that converts mol GSH to GSSG in the presence of hydrogen peroxide/min). 2.9.4. Glutathione reductase (GR) Glutathione reductase uses NADPH to convert oxidized glutathione (GSSG) to the reduced form (GSH). Its activity was determined by the method of Staal et al. [41]. Briefly, the assay mixture containing 0.2 ml of cell extract, 1.5 ml of sodium phosphate buffer, pH 7.0, 0.5 ml of 25 mM EDTA, 0.2 ml of 12.5 mM oxidized glutathione, and 0.1 ml of 3 mM NADPH were added and the decrease in absorbance at 340 nm compared for 1 min against blank containing all the components except cell extract in a spectrophotometer was measured. Activity was expressed as nmol of NADPH oxidized/min/mg protein. One unit of GR activity was defined as the amount of the enzyme that catalyzes the oxidation of mol of NADPH per minute. 2.9.5. Glutathione-S-transferase (GST) The activity of GST was assayed by the method of Habig et al. [42]. To 0.4 ml potassium phosphate buffer (0.5 mol/l; pH 6.5), 50 l of cell extract, 1.2 ml water and 0.1 ml CDNB (1-chloro-2,4dinitrobenzene, 30 mmol/l) conjugate were added and incubated in a water bath at 37 ◦ C for 10 min. After incubation, 0.1 ml of reduced glutathione (30 mmol/l) was added. The change in optical density was measured against blank at 340 nm at 30 s interval. Activity of GST was expressed as U/mg protein (1 U is the amount of enzyme that conjugates mol of 1-chloro-2,4-dinitrobenzene with GSH/min). 2.10. Assay of Sertoli cell enzymes 2.10.1. -GT Activity was determined by using l-␥-glutamyl-p-nitroanilide as substrate [43]. The colorless substrate exhibits maximum absorbance at 315 nm, while free p-nitroaniline is yellow and exhibits high absorbance between 350 and 420 nm. Enzymatic release of p-nitroaniline was followed at 410 nm, at which wavelength the substrate exhibits no absorbance. Determination of enzymatic activity was performed by adding 0.1 ml of cell extract to 0.9 ml of a solution (at 37 ◦ C) containing l-␥-glutamyl-pnitroanilide (5 mol), magnesium chloride (10 mol) and Tris–HCl buffer (pH 9.0; 100 mol). After 3–5 min of incubation, the reaction was terminated by addition of 2 ml of 1.5N acetic acid, and the absorbance was measured at 410 nm against a reference solution containing the same components except that the enzyme was added after addition of acetic acid. Enzymatic activity was determined by direct spectrophotometric measurement. The rate of generation of p-nitroaniline was found to be linear over that time. The activity of the enzyme was expressed as mol of p-nitroaniline formed during 1 min of incubation per mg of protein (1 U of enzyme activity was defined as mol of p-nitroaniline formed per minute per milligram of protein (mol/min/mg protein). 2.10.2. ˇ-Glucuronidase -Glucuronidase activity was determined by measuring the absorbance at 540 nm of phenolphthalein released from the substrate phenolphthalein glucuronide by the spectrophotometric method of Fishmann [44]. Total reaction volume was 150 l. The reaction mixture contained 50 l of reagent A (75 mM potassium phosphate buffer, with 1.0% (w/v) bovine serum albumin, pH 6.8 at 37 ◦ C), 25 l of reagent B (3.0 mM phenolphthalein glucuronide
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Table 1 Effect of LPS and lipoic acid on Sertoli cells viability.
Cell viability (%)
Group I control
Group II (LA)
Group III (LPS)
Group IV (LA + LPS)
91.33 ± 1.53
94.33 ± 1.15
90.67 ± 2.08
93.67 ± 0.58
Values are expressed as mean ± SD (n = 3 in each group). Comparisons are made between; Group I and Groups II–IV.
substrate solution), mix by inversion and equilibrate to 37 ◦ C. Then 10 l of cell extract were added and incubate at 37 ◦ C for exactly 30 min. The reaction was stopped by adding 500 l of 200 mM glycine buffer solution, pH 10.4. One unit of activity was defined as mol of phenolphthalein liberated per minute per milligram of protein (mol/min/mg protein). 2.10.3. Non-enzymatic antioxidant assay Total reduced glutathione (GSH) was estimated by the method of Sedalk and Lindsay [45], where the absorbance was measured at 412 nm. The amount of GSH was determined from a standard curve, under the same conditions, with a standard solution of GSH. GSH concentration was expressed as nmol/mg protein. Ascorbic acid (vitamin C) was assayed by the method of Omaye et al. [46]. Vitamin C was oxidized by copper to form dehydroascorbic acid which reacts with 2,4-dinitrophenylhydrazine to form the derivative bis2,4-dinitrophenylhydrazone. 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. The concentration of vitamin C was expressed as g/mg protein.
Fig. 1. Effect of lipopolysaccharide and lipoic acid on hydrogen peroxide production in adult rat Sertoli cells. Group I incubated with water (vehicle) throughout the experiment, served as a control. Group II was incubated with LA (100 M) alone for 24 h. Group III was treated with LPS (50 g/ml) alone for 12 h. Group IV was incubated (pretreatment) with (100 M) for 24 h then treated with LPS (50 g/ml) for 12 h. Values are mean ± SD (n = 3). Statistical analysis (ANOVA) for differences from corresponding control: comparisons were made between: a Group I and Groups II–IV; b Group III and Group IV. The symbols represent statistical significance from control where * P < 0.05; *** was a P < 0.001.
3.2. Lipid peroxidation (LPO) and hydrogen peroxide (H2 O2 ) production
2.11. Statistical analysis Results were expressed as mean ± standard deviation (SD), n = 3 (three independent culture preparations, each in triplicate). Differences in mean were analyzed by one-way analysis of variance (ANOVA). If F-value was significant, the data were subjected to Tukey–Kramer multiple comparison test. P < 0.05 was considered statistically significant. 3. Results
LPS-treated cells (Group III) showed increase in the amount of H2 O2 (96.5%), but not after LA pretreatment (Group IV; Fig. 1). There was also an abnormal increase of LPO in the LPS-treated Sertoli cells. The Group III cells displayed a 69% rise in basal LPO in cellular protein, as well as 53% in LPO following addition of the exogenous inducer ferrous sulphate compared to the control Group 1 (Table 1). LA pretreatment (Group IV) suppressed the increase in LPO induced by LPS (P < 0.05) (Table 2).
This study reported that at doses up to 500 g/ml for 24 h, LPS stimulated activin A and IL-1␣ (pro-inflammatory cytokine) secretion and inhibited inhibin B secretion by adult Sertoli cell cultures, and a maximum stimulatory effect was observed at a dose below 25 g/ml. Moreover, this study presented that higher doses of LPS (>500 g/ml) were toxic to the Sertoli cells, as indicated by the loss of cellular processes, rounding and detachment of the cells in culture.
3.3. Non-enzymatic antioxidants
3.1. Cell viability
3.4. Non-enzymatic antioxidants
The results showed that LPS did not significantly affect Sertoli cell viability in any of the experimental groups under our experimental conditions (Table 1).
Changes in Sertoli cell non-enzymatic antioxidants were shown in Table 3. LPS-treated cells (Group III) demonstrated a decrease in the concentration of GSH and antioxidant vitamin C (P < 0.001). LA
3.3.1. Lactate production The Sertoli cellular lactate concentration was significantly decreased (43%) in LPS-treated cells (Group III) when compared to control; however, supplementation of lipoic acid showed a significant increase in lactate concentration when compared to LPS-treated cells (P < 0.001) (Fig. 2).
Table 2 Effect of LPS and lipoic acid on lipid peroxidation in adult rat Sertoli cells. Lipid peroxidation
Group I control
Group II (LA)
Group III (LPS)
Group IV (LA + LPS)
Basal FeSO4 -induced
2.33 ± 0.21 7.6 ± 0.44
2.23 ± 0.21 7.9 ± 0.17
3.93 ± 0.15a , *** 11.6 ± 0.36a , ***
2.83 ± 0.15a , * , *** 8.1 ± 0.2b , ***
Values are expressed as mean ± SD (n = 3 in each group). Units—LPO: nM of MDA equivalent formed/mg protein. a Comparisons are made between; Group I and Groups II–IV. b Comparisons are made between; Group VIII and Group IV. * The symbols represent statistical significance from control: P < 0.05. *** The symbols represent statistical significance from control: P < 0.001.
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Fig. 2. Effect of lipopolysaccharide and lipoic acid on lactate concentration in adult rat Sertoli cells. Group I incubated with water (vehicle) throughout the experiment, served as a control. Group II was incubated with LA (100 M) alone for 24 h. Group III was treated with LPS (50 g/ml) alone for 12 h. Group IV was incubated (pretreatment) with (100 M) for 24 h then treated with LPS (50 g/ml) for 12 h. Values are mean ± SD (n = 3). Statistical analysis (ANOVA) for differences from corresponding control: comparisons were made between: a Group I and Groups II–IV; b Group III and Group IV. The symbols represent statistical significance from control where *** was a P < 0.001.
pretreatment maintained GSH and vitamin C concentrations near normal amounts. 3.5. Enzymatic antioxidants
Fig. 3. Effect of lipopolysaccharide and lipoic acid on ␥-GT (A) and -glucuronidase activity (B) in adult rat Sertoli cells. Group I incubated with water (vehicle) throughout the experiment, served as a control. Group II was incubated with LA (100 M) alone for 24 h. Group III was treated with LPS (50 g/ml) alone for 12 h. Group IV was incubated (pretreatment) with (100 M) for 24 h then treated with LPS (50 g/ml) for 12 h. Values are mean ± SD (n = 3). Statistical analysis (ANOVA) for differences from corresponding control: comparisons were made between: a Group I and Groups II–IV; b Group III and Group IV. The symbols represent statistical significance from control where *** was a P < 0.001.
Table 4 showed the activities of the antioxidant enzymes in Sertoli cell homogenate. LPS-treatment (Group III) decreased the specific activities of SOD (66%), CAT (39%), GPX (49%), GR (51%) and GST (42%) in comparison to control activities (Group I). All the LPSinduced alterations in the antioxidant enzymatic activities were effectively prevented by LA pretreatment. Moreover, LA-treated cells showed enhanced antioxidant capacity (Group II).
4. Discussion 3.6. Sertoli cell enzymes Bacterial infection of the genital tract may originate from the urinary tract or can be sexually transmitted [47,48]. This pathological infection will create an acute inflammatory response with an influx of leukocytes into the genital tract and a resulting increase in oxygen and oxygen-derived oxidant (ROS) production which may lead to oxidative stress [49–51]. These ROS include oxygen
The activities of ␥-GT and -glucuronidase were significantly decreased in LPS-treated cells (Group III) by 48% and 35% respectively compared to control (Group I). The activity of these enzymes was normalized by comparison to the LA pretreatment (Group IV, Fig. 3). Table 3 Effect of LPS and lipoic acid on non-enzymic antioxidants in adult rat Sertoli cells. Non-enzymic antioxidant
Group I control
Group II (LA)
Group III (LPS)
Group IV (LA + LPS)
GSH (nmol/mg protein) Vitamin C (g/mg protein)
11.47 ± 0.25 1.2 ± 0.1
12.5 ± 0.4 * 1.22 ± 0.01
5.57 ± 0.35 0.61 ± 0.03a , ***
11.03 ± 0.32b , *** 1.03 ± 0.06a , * , b , ***
a,
a , ***
Values represent mean ± SD (n = 3 in each group). a Comparisons are made between; Group I and Groups II–IV. b Comparisons are made between; Group VIII and Group IV. * The symbols represent statistical significance from control: P < 0.05. *** The symbols represent statistical significance from control: P < 0.001.
Table 4 Effect of LPS and lipoic acid on the activities of Sertoli cells antioxidant enzymes. Enzymatic antioxidants
Group I control
SOD CAT GPX GR GST
7 39.67 73 0.45 0.045
± ± ± ± ±
0.2 1.53 3 0.03 0.001
Group II (LA) 7.3 40.2 76.33 0.47 0.046
± ± ± ± ±
0.3 2.43 2.08 0.03 0.002
Group III (LPS) 2.37 24.3 37.33 0.22 0.026
± ± ± ± ±
0.15a , *** 1.4a , *** 2.52a , *** 0.03a , *** 0.003a , ***
Group IV (LA + LPS) 6.83 37.6 70.77 0.43 0.044
± ± ± ± ±
0.45b , *** 1.45b , *** 1.06b , *** 0.01b , *** 0.002b , ***
Values are expressed as mean ± SD (n = 3 in each group). Units—SOD: units/mg protein (1 U = amount of enzyme that inhibits the auto-oxidation of pyrogallol by 50%); CAT:.M of H2 O2 consumed/min/mg protein; GPX: M of reduced glutathione consumed/min/mg protein; GR: M of NADPH oxidized/min/mg protein; GST: M of 1-chloro-2,4dinitrobenzene-GSH conjugate formed/min/mg protein; ␥-GT: M of p-nitroaniline formed/min/mg protein. a Comparisons were made between: Group I and Groups II–IV. b Comparisons were made between: Group III and Group IV. *** The symbols represent statistical significance from control: P < 0.001.
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free radicals such as superoxide, hydroxyl, peroxyl, alkoxyl and hydroperoxyl radicals which cause tissue and cellular damage by a variety of mechanisms including DNA damage, lipid peroxidation, protein peroxidation, depletion of antioxidant enzymes and non-enzymatic antioxidant molecules. Evidence has implicated oxidative stress as an etiological factor in the development of male infertility [52]. In addition, Amin [53] reported that ROS are involved in a variety of patho-physiological conditions of testes. Sertoli cells create a microenvironment that protects seminiferous tubules from autoantigens and invading pathogens [54]. Previous studies have demonstrated that Sertoli cells produce the pro-inflammatory cytokines IL-1␣ and IL-6 and their secretion is stimulated by LPS, through stimulation of Toll-like receptors (TLRs) which play crucial roles in mediating innate and adaptive immunity [54]. Here, the protective effects of LA were shown. ␣-Lipoic acid is a racemic mixture R(+)-␣-LA is a cofactor of the pyruvate dehydrogenase and ␣-dehydrogenase complexes. Moreover, enantiomers of LA and their reduced form (dihydrolipoic acid) act as extraand intracellular redox couples and powerful lipophilic free radical scavengers. LA is highly reactive with free radicals and capable of increasing concentrations of GSH [55]. It has been shown that LA reduces oxidative stress in healthy adults and diabetic patients by decreasing significantly lipid peroxidation [55]. Therefore, the mechanism of LA protection for Sertoli cells can be inferred. In the present study, the decrease in Sertoli cellular lactate in LPS-treated cells may be due to oxygen toxicity and cytotoxicity through hydrogen peroxide that alters normal cellular metabolic function leading to a decrease in glycolysis. Several studies have indicated that lactate represents a preferential energy substrate for germ cells [56–58]. Lactate is produced in Sertoli cells from pyruvate following lactate dehydrogenase A (LDHA) action in Sertoli cells and is transported across the plasma membrane to the germ cells by specific proton/monocarboxylate transporters (MCTs) [59]. Lactate is then converted into pyruvate in germ cells following lactate dehydrogenase C (LDHC) action [60]. LPS-induced inflammation had a marked oxidative impact as evidenced by the significant increase of LPO and H2 O2 concentration. This change might have resulted from increased production of ROS and/or a decrease in antioxidant status. The high concentrations of polyunsaturated fatty acids in Sertoli cells caused susceptibility to lipid peroxidative degradation. The increased LPO due to LPS treatments was a consequence of impaired antioxidant enzyme activities and depletion in GSH and vitamin C concentrations. LPS-treated Sertoli cells may be more susceptible to LPO in the presence of promoter like ferrous sulphate. The elevated amounts of lipid peroxides that resulted from increased free radical formation might provide a biochemical mechanism for inflammation-associated testicular side effects. The prooxidant-antioxidant balance in LPS-induced inflammation was shifted toward prooxidants; hence, exogenous administration of a potential antioxidant such as LA was expected to normalize the prooxidant-antioxidant status. The ability of LA or its reduced form to scavenge free radicals may have restricted the production of toxic lipid peroxides and so reduced cellular need for enzymatic antioxidants. ROS enzymatic scavengers like SOD, CAT, GPx, GST and GR and antioxidants like GSH and vitamin C may protect cells from the injurious effects of ROS [61,15]. The decrease in activities of enzymatic antioxidants in LPS-induced inflammation in Sertoli cells might have increased LPO and ROS production. A decrease in SOD activity has been shown to increase the concentration of superoxide anions, which is known to inactivate GPx [62]. Equally, when GPx fails to eliminate H2 O2 from the cell the accumulated H2 O2 has been shown to cause inactivation of SOD [62]. Thus the balance of enzymes and pathways may be essential defenses against the superoxide anion and peroxides generated in
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the Sertoli cells. GST is inactivated either due to excessive production of H2 O2 [63] and/or reduced GSH amounts [63] as observed here. Thiols are the major components of cellular antioxidant system that play an important role as antioxidant against ROS and free radicals. GSH and its oxidized form glutathione disulphide constitute a major redox buffer system of the cell [64]. GSH is the major intracellular non-protein sulphydryl and its depletion increased the susceptibility of cells to oxidative stress [65]. Further, the decreased GSH content may explain the decreased concentration of vitamin C, which enters the cells mainly in the oxidized form where it is reduced by GSH [66]. The decrease in the concentration of this vitamin may have serious consequences as, in addition to antioxidant function, it plays a role in regenerating other antioxidants [66]. In summary, here LPS-induced inflammation in Sertoli cells induced significant oxidative stress that was associated with decreased enzymatic and non-enzymatic antioxidant molecules and the prooxidant-antioxidant balance was shifted towards prooxidants. Lipoic acid supplementation reduced oxidative stress in Sertoli cells treated with LPS by alleviating LPO through scavenging of free radicals or by enhancing the activities of antioxidant enzymes as well as the concentrations of vitamin C and GSH which then detoxify free radicals. Conflict of interest No conflicts of interest. References [1] M.E. Cutolo, M.K. Chan, H.Z. Movat, Sex hormone status of patients with rheumatoid arthritis: evidence of low serum concentrations at baseline and after human chorionic gonadotrophin stimulation, Arthritis Rheum. 31 (1988) 1314–1317. [2] J.P. Buch, S.K. Havlovec, Variation in sperm penetration assay related to viral illness, Fertil. Steril. 55 (1999) 844–846. [3] M. Shekarriz, A.J. Thomas, A. Agarwal, Incidence and level of seminal reactive oxygen species in normal men, Urology 45 (1995) 103–107. [4] A. Agarwal, S. Prabakaran, S. Allamanen, What an andrologist/urologist should know about free radicals and why, Urology 67 (2006) 2–8. [5] A. Agarwal, K. Makker, R. Sharma, Clinical relevance of oxidative stress in male factor infertility: an update, Am. J. Reprod. Immunol. 59 (2008) 2–11. [6] J.P. Buch, D.J. Lamb, L.I. Lipshultz, R.G. Smith, Partial characterization of a unique growth factor secreted by human Sertoli cells, Fertil. Steril. 49 (1988) 658–665. [7] A.R. Bellve, W. Zheng, Growth factors as autocrine and paracrine modulators of male gonadal functions, J. Reprod. Fertil. 85 (1989) 771–793. [8] M. Dym, D.W. Fawcett, The blood–testis barrier in the rat and the physiological compartmentation of the seminiferous epithelium, Biol. Reprod. 3 (1970) 308–326. [9] W.Y. Lui, D. Mruk, W.M. Lee, C.Y. Cheng, Sertoli cell tight junction dynamics: their regulation during spermatogenesis, Biol. Reprod. 68 (2003) 1087–1097. [10] B.P. Setchell, The testis and tissue transplantation: historical aspects, J. Reprod. Immunol. 18 (1990) 1–8. [11] A. Steinberger, G. Klinefelter, Sensitivity of Sertoli and Leydig cells to xenobiotics in in vitro models, Reprod. Toxicol. 7 (1993) 23–37. [12] G. Krishnamoorthy, P. Murugesan, R. Muthuvel, D.N. Gunadharini, A.R. Vijayababu, A. Arunkumar, P. Venkataraman, M.M. Aruldhas, J. Arunakaran, Effect of Aroclor 1254 on Sertoli cellular antioxidant system, androgen binding protein and lactate in adult rat in vitro, Toxicology 212 (2005) 195–205. [13] S. Srivastava, P.K. Seth, S.P. Srivastava, Effect of styrene administration on rat testis, Arch. Toxicol. 63 (1989) 43–46. [14] A.J. Grootegoed, P. Jansen, H.J. Vander Molen, The role of glucose, pyruvate and lactate in ATP production by rat spermatocyte and spermatid, Biochem. Biophys. Acta 762 (1984) 248–256. [15] F. Bauché, M.H. Fouchard, B. Jégou, Antioxidant system in rat testicular cells, FEBS Lett. 349 (1994) 392–396. [16] L. Packer, E.H. Witt, H.J. Tritschler, Alpha-lipoic acid as a biological antioxidant, Free Radic. Biol. Med. 19 (1995) 227–250. [17] K. Jacob, M.J. Periago, V. Böhm, G.R. Berruezo, Influence of lycopene and vitamin C from tomato juice on biomarkers of oxidative stress and inflammation, Br. J. Nutr. 99 (2008) 137–146. [18] S. Abe, Y. Tanaka, N. Fujise, T. Nakamura, H. Masunaga, T. Nagasawa, M. Yagi, An antioxidative nutrient-rich enteral diet attenuates lethal activity and oxidative stress induced by lipopolysaccharide in mice, JPEN J. Parenter. Enteral Nutr. 31 (2007) 181–187. [19] G.P. Biewenga, G.R. Haenen, A. Bast, The pharmacology of the antioxidant lipoic acid, Gen. Pharmacol. 29 (1997) 315–331.
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