- Email: [email protected]
Methoxychlor induces apoptosis via mitochondria- and FasL-mediated pathways in adult rat testis
Chemico-Biological Interactions 185 (2010) 110–118
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Methoxychlor induces apoptosis via mitochondria- and FasL-mediated pathways in adult rat testis S. Vaithinathan, B. Saradha, P.P. Mathur ∗ Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Pondicherry 605 014, India
a r t i c l e
i n f o
Article history: Received 31 December 2009 Received in revised form 3 March 2010 Accepted 5 March 2010 Available online 12 March 2010 Keywords: Methoxychlor Testis Apoptosis Caspase NF-B FasL
a b s t r a c t In the past few years, there has been much concern about the adverse health effects of environmental contaminants in general and organochlorine in particular. Studies have shown the repro-toxic effects of long-term exposure to methoxychlor, a member of the organochlorine family. However, the insight into the mechanisms of gonadal toxicity induced by methoxychlor is not well known. In the present study we sought to elucidate the mechanism(s) underpinning the gonadal effects within hours of exposure to methoxychlor. Experimental rats were divided into six groups of four each. Animals were orally administered with a single dose of methoxychlor (50 mg/kg body weight) and killed at 0, 3, 6, 12, 24, and 72 h post-treatment. The levels and time-course of induction of apoptosis-related proteins like cytochorome C, caspase 3 and procaspase 9, Fas–FasL and NF-B were determined to assess sequential induction of apoptosis in the rat testis. DNA damage was assessed by TUNEL assay and flowcytometry. Administration of methoxychlor resulted in a significant increase in the levels of cytosolic cytochrome c and procaspase 9 as early as 6 h following exposure. Time-dependent elevations in the levels of Fas, FasL, pro- and cleaved caspase 3 were observed. The DNA damage was measured and showed time-dependent increase in the TUNEL positive cells, and also by flowcytometry of testicular cells. The study demonstrates induction of testicular apoptosis in adult rats following exposure to a single dose of methoxychlor. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In the recent years increased incidence of reproductive disorders has elevated concerns about the adverse effect of endocrinedisrupting chemicals on reproductive health in humans and wild life. Most of the endocrine disruptors released into the environment show estrogenic properties and exert their effects by mimicking endogenous hormones thereby leading to the disruption of endocrine mechanisms [1]. Methoxychlor is a broad spectrum chlorinated insecticide which is used to control insects on agricultural crops, livestock, animal feeds, barns, home, gardens and on household pets [2]. Thus, humans as well as wildlife species are exposed to methoxychlor easily. Methoxychlor acts as a proestrogen but it can be metabolized by the liver into mono-, bis-, tris-hydroxy and Catechol-M, which are estrogenic in nature than parental compound [3,4]. The most active estrogenic metabolite is bis-hydroxy methoxychlor(2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) [2,5]. Recent findings reveal that methoxychlor reduces fertility in male rats by reducing the weight of seminal vesicle, serum testosterone and dehydroepiandrosterone at a dose level of 200 mg/kg
∗ Corresponding author. Tel.: +91 413 2655212; fax: +91 413 2655211. E-mail address: [email protected] (P.P. Mathur). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.03.014
body weight for 7 days [6], ovarian atrophy and decreases the ability of ovarian cells to synthesize and secrete hormones in female rats [7,8]. The metabolite 2,2-bis-(p-hydroxyphenyl)-1,1,1trichloroethane (HPTE) is known to act as estrogen receptor ER ␣ agonist and ER  antagonist in human hepatoma cells [9]. Exposure to methoxychlor at doses of 0, 5, 50, and 150 mg/kg/day during the perinatal/juvenile period, reduced the spermatogenesis of males as adults by reducing their Sertoli cell number [10]. Oral exposure to methoxychlor caused atrophy of male sexual organs in rats at the dosage of 100 and 200 mg/kg/day for 70 days [11]. Methoxychlor exposure to pregnant female rats from embryonic days 8 to 15 at the dosage of 100 and 200 mg/kg/day showed an increase in spermatogenic cell apopotis and decreased sperm number and motility in adult animals of F1 and F2 generation [12,13]. Studies from our laboratory have exhibited a state of oxidative stress induced in adult rat testis following short- and longterm exposure of methoxychlor. Oxidative stress is induced by promoting lipid peroxidation, and a decrease in the activity of antioxidant enzymes in testis and epididymis [14–16]. Recently, we have demonstrated a transient inhibitory effect of methoxychlor (50 mg/kg body weight) on testicular steroidogenic enzymes, 5 3-hydroxysteroid dehydrogenase and 5 17-hydroxysteroid dehydrogenase within 6–12 h of exposure and a possible role of hydrogen peroxide (H2 O2 ) in mediating these effects was suggested [17]. Methoxychlor showed alteration in the levels of heat shock
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118 Table 1 Antibodies used in the present study. Vendor
Antibody
Host species
Catalog no.
Santa Cruz Biotechnology Santa Cruz Biotechnology Santa Cruz Biotechnology Santa Cruz Biotechnology Santa Cruz Biotechnology
Caspase 3 Caspase 9 Fas Fas-L NF-кB
Goat Rabbit Rabbit Rabbit Rabbit
SC-1225 SC-7885 SC-7886 SC-834 SC-7151
protein and clusterin accompanied by an induction of oxidative stress in the testis at a same dose [18]. Cell death by apoptosis is a part of normal development and maintenance of testicular homeostasis. During various stages of spermatogenesis, an adequate amount of germ cells are eliminated via the process of apoptosis in order to maintain a precise germ cell population in compliance with the supportive capacity of the Sertoli cells. The two divergent pathways of testicular apoptosis namely, the receptor-mediated Fas/FasL pathway and the mitochondrial-mediated pathway play an important role in maintaining the germ cell population [24]. Inappropriate activation of apoptosis could enormously jeopardize spermatogenesis and hence fertility. Reactive oxygen species (ROS) is considered a potential signal for apoptosis. Elevated levels of ROS can cause oxidation of the mitochondrial pores thereby disrupting the mitochondrial membrane potential and releasing cytochrome c thereby activating the mitochondrial-mediated pathway of apoptosis. In addition, ROS have been shown to induce the expression of Fas receptor and ligand stimulating the Fas/FasL-mediated apoptotic signal transduction pathway. Several environmental disruptors are known to inappropriately activate apoptosis in testicular locale by increasing the levels of ROS [29,35]. Taking a lead from these reports and our previous studies, we sought to investigate whether short-term treatment with a low dose of methoxychlor would alter the levels of apoptosis-related proteins in adult rat testis. The aim of the present study was to determine the effects of methoxychlor on testicular apoptosis and to investigate FasL-dependent pathway. We investigated the expressions of Fas, FasL, and activation of NF-B and to find whether caspase family members play an important role in apoptosis, it is also of interest to determine the expression levels of caspase 3 and procaspase 9 in methoxychlor-induced apoptosis.
111
galore, India) and water ad libitum. Seven days prior to the start of the experiments, rats were handled daily for 5 min to acclimatize them to human contact and minimize their physiological responses to handling. The experiments were carried out in accordance with the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Government of India (CPCSEA 2003) and were approved by the Institutional Animal Ethics Committee of Pondicherry University. Methoxychlor was dissolved in olive oil and administered (50 mg/kg body weight) to all treatment groups through oral gavage. Each group consisted of four animals. After treatment with methoxychlor for various times (0, 3, 6, 12, 24, and 72 h) animals were killed by cervical dislocation. Testes were collected, cleared of the adhering tissues, weighed and stored at −80 ◦ C until analysis.
2. Materials and methods 2.1. Chemicals Methoxychlor(1,1,1 trichloro-2,2-bis(4-methoxyphenyl) ethane, ∼95% pure) was purchased from Sigma Chemical Company (St. Louis, MO, USA). Details on primary antibodies used in this study are listed in Table 1. Goat anti-rabbit IgG and rabbit anti-goat IgG conjugated to FITC, goat anti-rabbit IgG conjugated to Cy3TM , horseradish peroxidase-conjugated goat anti-rabbit IgG and rabbit anti-goat IgG were obtained from Bangalore Genei (Bangalore, India). TUNEL kit was purchased from Promega, USA. All other chemicals used were of analytical grade and were obtained from local commercial sources. 2.2. Experimental design Adult male rats (80–90 days), of Wistar strain procured from an authorized rat vendor (M/S Raghavendra Enterprises, Bangalore, India), were housed in polypropylene cages and maintained at 22–25 ◦ C under a well-regulated light and dark (12:12 h) schedule in the animal house at Pondicherry University. The animals were provided with standard rat chow (Sai Durga Feeds and Foods, Ban-
Fig. 1. Effect of methoxychlor in the levels of cytochrome c, procaspase-9 and -3 in the testis of methoxychlor-treated rats over different time points. Methoxychlor (50 mg/kg) was administered to adult male rats (n = 4) which were killed at specific time points thereafter. (A) Immunoblots showing significant increase in the cytosolic levels of cytochrome c (11 kDa) from 6 h post-treatment. Immunoblot of procaspase 9 (37 kDa) shows a parallel increase from 6 h onwards. Maximum changes in the levels of cytochrome c and procaspse 9 are observed at 24 and 72 h post-treatment. The level of cytochrome c declines at 72 h, yet remains significantly higher than control. A sequential increase in the levels both procaspase 3 (32 kDa) and the cleaved fragment (p17) is observed from 6 to 72 h post-exposure. Immunoblot showing sequential increase in the levels of Fas (48 kDa) and FasL (30 kDa) from 6 to 24 h post-treatment. Immunoblot of NF-B shows time-dependent decrease in the cytosolic levels of NF-B p65 with a maximum decline at 12 h post-exposure. By 72 h, the levels of NF-B p65 in the cytosolic extract increase, yet remains significantly higher than control. Actin blot shows equal protein loading. Each of these depicted blots is representative of four experiments. (B) Graph representing densitometrically scanned results corresponding to the blots depicted in A. Each data point represents the average value from four independent experiments normalized against the control (0 h). The control was arbitrarily set at 1. Asterisk indicates values significantly different p < 0.05 as compared with control.
112
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
Fig. 2. Co-localized expression of caspase 3 and Fas in the testis of methoxychlor-treated rats; (a, d, g, and j) visualization of caspase 3 staining; (b, e, h, and k) visualization of Fas staining; (c, f, i, and l) visualization of overlap of Fas and caspase-3 staining. Fas and caspase 3 is localized to peritubular germ cells (spermatogonia and spermatocytes) and their coexistence is visualized (c, f, i, and l) when green (caspase 3) and red fluorescence (Fas) are superimposed (yellow to orange). A time-dependent increase in the expression of caspase 3 and Fas in the testis of methoxychlor-treated rats may be noted. The bar represents 50 m. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)
2.3. Immunoblot analysis of caspase-3, -9, cytochrome c, Fas, FasL and NF-B Testes lysates were prepared in the lysis buffer (50 mM Tris, pH 7.4 containing 0.15 M NaCl, 10% glycerol (v/v), 1% NP-40 (v/v), 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 1 mM EDTA, 150 M bestatin, 1 M leupeptin and 1 M aprotinin) using a tissue:buffer ratio of 1:5. After homogenization, samples were first centrifuged at 1000 × g for 2 min to remove large tissue fragments and then centrifuged again at 10,000 × g for 30 min. The supernatants (cytoplasmic extract) were collected and stored at −70 ◦ C. The protein concentration was determined by Lowry’s method [19]
and equal quantities of protein were loaded per lane and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis [Mini Protean II System, Bio-Rad] as described by Laemmli [20]. Electrophoresis was performed at 75 V and the resolved proteins were electrophoretically transferred onto a nitrocellulose membrane (NYTRAN, Keene, NH, USA) in the transfer buffer (0.2 mol/L glycine, 25 mM Tris, and 20% methanol). Successful transfer was confirmed by Ponceau S staining of the blots. The membranes were incubated in a blocking buffer (phosphate-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dry milk powder) for 5 h at room temperature, followed by incubation in respective primary antibodies. Anti-caspase 3 was diluted at 1/100, and anti-
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
caspase 9, anti-cytochrome c, anti-Fas, anti-FasL and anti-NF-B were diluted at 1/200. The incubation with the primary antibodies was carried out overnight at 4 ◦ C. The following day, blots were washed in phosphate-buffered saline and incubated for 1 h at room temperature with either horseradish peroxidase-conjugated antirabbit IgG or anti-goat IgG (1:1000 dilution). Immunodetection of proteins was revealed by using tetramethylbenzidine/hydrogen peroxide (TMB/H2 O2 ) (Bangalore Genei, Bangalore, India) as a substrate and resulting immnunospecific bands were quantified by densitometry. 2.4. Immunofluorescent staining of Fas, FasL, caspase 3 and NF-B Rat testes were fixed according to Bouin’s method and embedded in paraffin and sectioned at a thickness of 5 m on poly-l-lysine-coated slides. After deparaffinization and rehydration, sections were washed in PBS (10 mM NaH2 PO4 , pH 7.4 at 22 ◦ C containing 0.15 M NaCl) and permeabilized in 0.2% Triton X100 (v/v). Sections were then blocked with 10% non-immune goat serum for 1 h. Sections were incubated with anti-FasL and anti-NFB (1:100 dilution each containing 1% non-immune goat serum) antibodies overnight at 4 ◦ C. After repeated rinsing with PBS, the sections were saturated with goat anti-rabbit IgG conjugated to FITC (1:100 dilution containing 10% non-immune goat serum) for 2 h in the dark. After being washed in PBS three to four times (5 min each), the slides were mounted and examined under the microscope (Olympus System, model CX 41RF, Japan). To determine whether Fas and caspase 3 were colocalized, the sections were sequentially incubated with appropriate combinations of antibodies. The tissue sections were incubated with primary antibody mixture, which consisted of rabbit anti-Fas and goat anti-caspase 3 (1:100 dilution each containing 1% nonimmune goat serum) overnight at 4 ◦ C. After repeated rinsing with PBS, the sections were incubated with secondary antibody mixture, which consisted of goat anti-rabbit IgG conjugated to Cy3TM and rabbit anti-goat IgG conjugated to FITC (1:100 dilution each containing 10% non-immune goat serum). After being washed in PBS three to four times (5 min each), the slides were mounted and examined under the microscope (Olympus System, model CX 41RF, Japan). 2.5. TUNEL assay Apoptotic cells were detected using the Dead End Colorimetric Apoptosis Detection System (Promega Corp., Madison, WI, USA). This system end-labels the fragmented DNA of apoptotic cells using a modified terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. It consists of incorporation of biotinylated nucleotides at the 3 -OH DNA ends using the terminal deoxynucleotidyl transferase, recombinant (rTDT) enzyme. Horseradishperoxidase-labeled streptavidin is then bound to these biotinylated nucleotides, which are detected using the peroxidase substrate, hydrogen peroxide and the stable chromogen diaminobenzidine (DAB). Briefly, the tissue sections were deparaffinized, rehydrated and fixed in 4% paraformaldehyde solution in PBS. Proteinase K (20 g/ml) treatment for 15 min was followed by a second fixation in 4% paraformaldehyde solution in PBS. The sections were incubated with equilibration buffer (200 mM potassium cacodylate, pH 6.6, 25 mM Tris–HCl, pH 6.6, 0.2 mM DTT, 0.25 mg/ml BSA, 2.5 mM cobalt chloride) for 10 min. Subsequently, the sections were allowed to react with rTDT reaction mixture (98 l equilibration buffer, 1 l biotinylated nucleotide mix and 1 l rTDT enzyme) for 1 h at 37 ◦ C in a humidified chamber. The slides were immersed in 2× SCC for 15 min to stop the reaction and the endogenous peroxidase were blocked by incubating the sections with 0.3% H2 O2 in PBS for 5 min. After
113
incubation with streptavidin–HRP solution for 30 min at room temperature, the slides were stained with DAB–H2 O2 in the dark. Positive nuclei stained dark-brown and were visualized under a light microscope. Negative controls with TUNEL assay were performed according to the instructions provided by the manufacturer. 2.6. Flowcytometry analysis Testicular suspensions were prepared by the following method. Briefly, the tunica albuginea was removed and the decapsulated testis was placed in ice cold nutrient medium (Dulbecco’s modified eagle medium) and minced gently with razor. Then the tissue was transferred to nutrient medium containing 0.25% collagenase (Type IA) (Sigma, St. Louis, USA) for 30 min in a shaking water bath at 32.5 ◦ C. The cell suspension was filtered through a 100-m and 40-m nylon mesh (BD Falcon, Bedford, USA) in order to discard tissue debris and to obtain single cell suspension, respectively. The suspension was centrifuged at 300 × g for 5 min and the pellet was washed twice with medium. One aliquot of each cell suspension was stained with 0.2% trypan blue for the determination of viability. The cells were fixed in ice cold 70% methanol and stored at 20 ◦ C for minimum 24 h. After fixation cells were incubated with 0.25% RNase (Bangalore Genei, India) (37 ◦ C, 20 min). After washing twice with PBS, DNA staining was carried out by Propidium Iodide (PI; Sigma, St. Louis, USA) solution (50 g/ml in PBS), and placed in the dark for 30 min at 4 ◦ C. Enumerations of cells on the basis of their DNA content were carried out in a Flow Cytometer (FACS Calibur, Becton-Dickinson, USA) equipped with air-cooled 15 mv Argon ion laser at 488 nm and the emitted light from cells was detected at 580 ± 30 nm on FL2. The DNA histograms were analyzed by Cell Quest (Becton-Dickinson, USA) software. 2.7. Statistical analysis Data were expressed as mean ± SD for four animals per group. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s post-test using SPSS (student version 7.5, SPSS Inc., UK). p < 0.05 was taken as statistically significant. Images were compiled using Adobe Photoshop (version 7.0, San Jose, CA). Densitometric scanning was performed using Gene tool (version 3.05, Synoptics Ltd., Cambridge, UK). 3. Results 3.1. Expression of cytochrome c, caspase 9 and caspase 3 proteins in methoxychlor-treated rats To assess the effect of methoxychlor on the mitochondriadependent cell death pathway, the levels of cytosolic cytochrome c, procaspase 9 and caspase 3 proteins were evaluated in adult rat testis. Western blot analysis revealed a significant elevation in the levels of cytosolic cytochrome c (11 kDa) and procaspase 9 (37 kDa) as early as 6 h following exposure to a single dose of methoxychlor (Fig. 1). Maximal changes in the levels of cytochrome c and procaspse 9 were observed at 12 and 72 h post-treatment. A time-dependent elevation in the levels of procaspase 3 (32 kDa) accompanied by an increase in the cleaved fragment (17 kDa) was observed following exposure to a single dose of methoxychlor (Fig. 1). 3.2. Effect of methoxychlor on the protein levels of Fas, FasL and NF-B Cytoplasmic extract of the testis was used to assess the effect of methoxychlor on Fas–FasL system by western blot analysis
114
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
Fig. 3. Immunofluorescent staining of FasL in the testis of methoxychlor-treated rats. Representative images for FasL immunoreactivity in control (a), 6 h (b), 12 h (d) and 72 h (f) post-treated groups. (c, e, and g) Magnified images of (b, d, and f), respectively. FasL is localized to the cytoplasmic extensions of Sertoli cells (arrows heads) and peritubular germ cells (asterisks). A time-dependent increase in the expression of FasL in the testis of rats exposed to a single dose of methoxychlor may be noted. The bar represents 50 m.
and to delineate the possible role of NF-B-mediated effects. A significant increase in the levels of Fas and FasL in a timedependent manner from 6 h onwards reaching a peak at 24 h in the methoxychlor-treated rats was observed (Fig. 1). On the contrary, a time-dependent decrease in the levels NF-B, with a significant decline at 12 and 24 h was resulted. However, the levels started recouping at 72 h post-treatment (Fig. 1). 3.3. Colocalized expression of caspase 3 and Fas Co-localization of Fas and Caspase 3 showed a time-dependent increase in the methoxychlor-treated rats. Fas and caspase 3 were localized in spermatogonia and spermatocytes during double immunofluorescent labeling, and their coexistence was visualized
Fig. 4. Immunofluorescent staining of NF-B p65 in the testis of methoxychlortreated rats. Representative images for NF-B p65 immunoreactivity in control (a), 6 h (c), 24 h (e) and 72 h (g) post-treated groups. (b, d, f, and h) Magnified image of (a, c, e, and g) respectively. A time-dependent increase in nuclear localization of NF-B p65 in spermatogonia (asterisks) and spermatocytes (arrow heads) with a maximal nuclear staining at 24 h post-exposure (e and f) may be noted. At 72 h, the nuclear staining is less pronounced (g and h). The bar represents 50 m.
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
115
Fig. 5. Methoxychlor-induced germ cell death in the testis of rats as revealed by TUNEL assay. Representative images for TUNEL staining in negative control (a), control (b), 6 h (c), 12 h (d) and 72 h (e) post-treatment groups. The TUNEL-positive cells (arrows) are found in the peripheral region near the basement membrane of the seminiferous tubules. A time-dependent increase in the number of apoptotic cells in the testis of methoxychlor-treated rats may be noted. The bar represents 50 m.
when green and red fluorescence were superposed (yellow to orange in Fig. 2).
labeled germ cells increased progressively. TUNEL-labeled germ cells were principally spermatogonia and spermatocytes in the methoxychlor-treated rats.
3.4. Immunolocalization of FasL and NF-B in testis of methoxychlor-treated rats
3.6. Flowcytometry analysis of apoptosis
Expression of FasL and NF-B protein in the methoxychlor exposed rat testis was examined by immunofluorescent microscopy. Cytoplasm of Sertoli cells and peritubular cells showed a FasL expression. In a time-dependent manner from 6 to 72 h immunoreactivity of FasL showed an increase in the testis (Fig. 3). In methoxychlor-treated rats NF-B p65 subunit changed from cytoplasm to nucleus of germ cells. This change in the localization of NF-B p65 subunit was observed from 3 h with maximal nuclear reactivity at 12 h post-expsoure (Fig. 4). However, at 72 h the nuclear localization of p65 subunit was less pronounced and only a few tubules displayed nuclear reactivity in germ cells.
Total germ cell number was determined using flowcytometric analysis of propidium iodide labeled cells (Fig. 6). We detected three major histogram peaks of DNA content, which represented M1 (elongating and elongated spermatid), haploid-M2 (round spermatid), diploid-M3 (spermatogonia, preleptotene primary and secondary spermatocytes) and tetraploid-M4 (spermatogonia, leptotene). Compared with control animals, 6 h post-treatment of methoxychlor showed a time-dependent increase in elongated cells (Figs. 6 and 7). A time-dependent decrease of diploid cells occurs in post-treatment of methoxychlor. These results correlate with the TUNEL results.
3.5. TUNEL assay
4. Discussion
In situ TUNEL analysis quantitative data from TUNEL-labeled germ cells are shown in Fig. 6. In the testes when compared to the control rats (3.25 ± 1.02), the percentage of TUNEL-labeled germ cells after a single dose of methoxychlor increased in the peripheral regions in time-dependent manner at 6 h (8.66 ± 2.10) and 72 h (21.98 ± 3.21) post-treatment (Fig. 5). The number of TUNEL-
The current study was conducted to interpret the testicular response to methoxychlor in adult male rats. Literature reveals metabolism and biotransformation of methoxychlor in rats and its elimination by the system within 48 h following the oral exposure [21,22]. Our previous studies have demonstrated the induction of oxidative stress and inhibition of steroidogenesis to a single expo-
116
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
sure of methoxychlor at 50 mg/kg body weight [17,18]. Knowledge on this terrain as well as the high vulnerability of the testis to methoxychlor prompted us to study the stress-mediated apoptosis following exposure to single dose of methoxychlor. The results of the present study indicate that methoxychlor, at a concentration of 50 mg/kg body weight, has the ability to induce apoptosis in testicular cells in vivo after 12 h of exposure. The present study sought to investigate the sequential changes in the levels of cytochrome c, procaspase 9 and caspase 3, Fas and FasL proteins in the testis of adult rats following exposure to a single dose of methoxychlor. There is a universal consent that male reproductive organs are particularly susceptible to the detrimental effects of reactive oxygen species (ROS) and lipid peroxidation, which lead to impaired fertility [23]. Reactive oxygen species (ROS) is considered a potential signal for apoptosis. There is evidence that ROS plays a role in the induction of both death receptor and mitochondrial-apoptotic pathways [24]. Particularly in the testes, the germ cells such as spermatozoa are highly receptive to ROS damage and damage from ROS-generated products due to the prevalence of oxidation-prone unsaturated fatty acids on their plasma membrane. Apoptosis involves a cascade of signals and there are different pathways involved in testicular cell apoptosis pertaining to specific treatments. Literature reveals as a consequence of ROS generation in cells, mitochondrial dysfunction should occur [25]. In the present study methoxychlor treatment showed a significant increase in the level of cytoplasmic cytochrome c at 6–24 h following exposure. A significant elevation of procaspase 9 was observed in 6 h of posttreatment. The changes in the levels of both proteins coincide at the same time points. The results indicate that cytochrome c acti-
vates procaspase 9, which in turn activates executioner caspases, leading to the intrinsic pathway of apoptosis. The maximal levels of cytochrome c and procaspase 9 coincide with our previous studies where the decrease in the antioxidant enzymes levels and accumulation of lipid peroxidation [18]. In this study, we observed a significant increase in the activation of caspase 3 followed by an elevation in cleaved caspase 3 from 6 h of post-treatment. Elevation in the level of cleaved caspase 3 after the release of cytochrome c accompanied by an increase in procaspase 9 suggest the activation of intrinsic pathway of apoptosis in the testis of treated rats. Methoxychlor-induced apoptosis in the testis may be caused by increased oxidative stress after the treatment of methoxychlor. The Fas system has been widely recognized as a key regulator of the apoptosis signal transduction pathways. Fas is a type I transmembrane receptor protein and Fas ligand (FasL) is a type II transmembrane protein. The ligation of FasL to Fas triggers an FasLmediated apoptotic death in a target cell leading to the activation of caspase 3 [26,27]. All the cells expressing both Fas and FasL do not undergo apoptosis under normal conditions, but when an external stimuli is induced then can become significantly sensitized to the Fas-mediated apoptosis [28]. Our findings revealed an evident induction of both Fas and FasL levels in testis after methoxychlor treatment, when in comparison to control groups. The highest expression of FasL protein was detected at 24 h post-treatment of methoxychlor, while the levels of Fas protein reached to the maximum at 24 h when the level of apoptosis also peaked. This result was in accordance to onset of FasL mRNA upregulation after Sertoli cell injury [29]. Immunofluorescent staining showed a timedependent expression of FasL in the Sertoli cells and spermatogonia
Fig. 6. Flow cytometric analysis of testicular cells from control and methoxychlor-treated rats. Four different cell populations corresponding to four different peaks such as elongating and elongated spermatid as M1, haploid cells as M2 (round spermatid), diploid cells like spermatogonia, preleptotene primary and secondary spermatocytes as M3 and tetraploid cells, spermatogonia, leptotene as M4 are observed in control group. There was a decrease in time-dependent manner in the amplitude of haploid cells in the methoxychlor-treated groups.
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
117
Fig. 7. Germ cells isolated from testes of control and methoxychlor-treated rats (live versus dead-arrow). Number of viable cells reduced time-dependent manner in methoxychlor-treated rats (a–d).
at 6–72 h post-treatment of methoxychlor. The result indicated that increased expression of FasL in Sertoli cells triggers apoptotic elimination of Fas-expressing germ cells, suggests that the upregulation of FasL expression in Sertoli cells and increased activation of caspase 3 may be associated with apoptosis in methoxychlor-treated rats. Co-localization of caspase 3 and Fas showed an increased expression of caspase 3 and Fas at 12–72 h of post-treatment which correlates with our earlier result acquired by western blot. This shows that both the pathways occur in same cell type in the methoxychlor-treated rats when compared to control animals. The results further confirm the involvement of Fas-mediated pathway in methoxychlor-induced apoptosis. Animals when exposed to specific testicular toxicants, such as 2,5-hexanedione or mono2-ethylhexyl phthalate, which target the germ cell and Sertoli cells and disrupt their functions, will lead to up-regulation of Fas and FasL in the injured cells, respectively. This up-regulation result in a Fas–FasL-mediated elevation of germ cell apoptosis [29–31]. The ligation of Fas to FasL could stimulate NF-B activation [32]. NF-B is a complex of p65 and p50 proteins and is found to be constitutively expressed in the nuclei of rat testicular cells. In normal cells, NF-B dimers are sequestered in the cytoplasm by inhibitory kappa B (IB). When it exposed to external signal degradation of IkB, free NF-B translocates to the nucleus. NF-B exerts both proand antiapoptotic effects in testis upon toxicant exposure [33]. Our study demonstrated that single dose exposure to methoxychlor could decrease the level of p65 subunit of NF-B in cytoplasmic homogenate of the testis at 6 h of post-treatment. The maximum reduction of NF-B occurs at 12 h and started to return normal at 72 h of post-treatment. Immunofluoresence results showed that nuclear translocation of NF-B in germ cells of methoxychlortreated rats from 6 h with a maximum of 24 h of post-treatment
which shows an apoptotic role of NF-B in testis. Both intrinsic and extrinsic pathways of apoptosis enter into a common downstream effector, caspase 3, which can induce DNA fragmentation by the activation of endonuclease. In this study, methoxychlor-induced apoptosis in male rat germ cells in a time-dependent manner. TUNEL-labeled germ cells induced by methoxychlor reached a maximum in number at 12 h after dosing. Cell death by apoptosis is a relatively rapid mechanism, and complete removal of the cell often occurs within hours from the tissue [34]. This has been confirmed by previous reports [35–37]. Thus, germ cell cytotoxicity exerts within a short period of methoxychlor treatment. Germ cell apoptosis in the rodent testes has also been observed as a part of normal spermatogenesis [38], which is increased by testicular toxicants. In addition, support to this TUNEL observation came from the fact that there was a time-dependent reduction of haploid cells as observed by flowcytometry. In conclusion, exposure to a single dose of methoxychlor induces apoptosis via mitochondria-mediated and FasL-dependent pathways in testis of adult rats. Conflict of interest None. Acknowledgements P.P. Mathur acknowledges the receipt of financial support from the Department of Science and Technology, Government of India under the projects (1) SP/SO/B-65/99 (2) DST-FIST and (3) Indian Council of Medical Research, New Delhi. B. Saradha acknowledges the Indian Council of Medical Research, New Delhi, India for Senior Research Fellowship. The authors also thank the staff of Bioinfor-
118
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
matics Center, Pondicherry University, Pondicherry for providing various facilities. References [1] B. Saradha, P.P. Mathur, Effect of environmental contaminants on male reproduction, Environ. Toxicol. Pharmacol. 21 (1) (2006) 34–41. [2] A.M. Cummings, Methoxychlor as a model for environmental estrogens, Crit. Rev. Toxicol. 27 (4) (1997) 367–379. [3] Y. Hu, D. Kupfer, Enantioselective metabolism of the endocrine disruptor pesticide methoxychlor by human cytochromes P450 (P450 s): major differences in selective enantiomer formation by various P450 isoforms, Drug Metab. Dispos. 30 (12) (2002) 1329–1336. [4] Y. Hu, D. Kupfer, Metabolism of the endocrine disruptor pesticidemethoxychlor by human P450 s: pathways involving a novel catechol metabolite, Drug Metab. Dispos. 30 (9) (2002) 1035–1042. [5] S.S. Dehal, D. Kupfer, Metabolism of the proestrogenic pesticide methoxychlor by hepatic P450 monooxygenases in rats and humans: dual pathways involving novel ortho ring-hydroxylation by CYP2B, Drug Metab. Dispos. 22 (6) (1994) 937–946. [6] E.P. Murono, R.C. Derk, Y. Akgul, In vivo exposure of young adult male rats to methoxychlor reduces serum testosterone levels and ex vivo Leydig cell testosterone formation and cholesterol side-chain cleavage activity, Reprod. Toxicol. 21 (2) (2006) 148–153. [7] E.M. Martinez, W.J. Swartz, Effects of methoxychlor on the reproductive system of the adult female mouse: 2. Ultrastructural observations, Reprod. Toxicol. 6 (1) (1992) 93–98. [8] W.J. Swartz, M. Corkern, Effects of methoxychlor treatment of pregnant mice on female offspring of the treated and subsequent pregnancies, Reprod. Toxicol. 6 (5) (1992) 431–437. [9] K.W. Gaido, S.C. Maness, D.P. McDonnell, S.S. Dehal, D. Kupfer, S. Safe, Interaction of methoxychlor and related compounds with estrogen receptor alpha and beta, and androgen receptor: structure-activity studies, Mol, Pharmacology 58 (4) (2000) 852–858. [10] R.E. Chapin, M.W. Harris, B.J. Davis, S.M. Ward, R.E. Wilson, M.A. Mauney, A.C. Lockhart, R.J. Smialowicz, V.C. Moser, L.T. Burka, B.J. Collins, The effects of perinatal/juvenile methoxychlor exposure on adult rat nervous, immune, and reproductive system function, Fundam, Appl. Toxicol. 40 (1) (1997) 138–157. [11] H.S. Bal, Effect of methoxychlor on reproductive systems of the rat, Proc. Soc. Exp. Biol. Med. 176 (2) (1984) 187–196. [12] M.D. Anway, A.S. Cupp, M. Uzumcu, M.K. Skinner, Epigenetic transgenerational actions of endocrine disruptors and male fertility, Science 308 (5727) (2005) 1466–1469. [13] A.S. Cupp, M. Uzumcu, H. Suzuki, K. Dirks, B. Phillips, M.K. Skinner, Effect of transient embryonic in vivo exposure to the endocrine disruptor methoxychlor on embryonic and postnatal testis development, J. Androl. 24 (5) (2003) 736–745. [14] C. Latchoumycandane, P.P. Mathur, Induction of oxidative stress in the rat testis after short-term exposure to the organochlorine pesticide methoxychlor, Arch. Toxicol. 76 (12) (2002) 692–698. [15] C. Latchoumycandane, P.P. Mathur, Effect of methoxychlor on the antioxidant system in mitochondrial and microsome-rich fractions of rat testis, Toxicology 176 (1–2) (2002) 67–75. [16] C. Latchoumycandane, K.C. Chitra, P.P. Mathur, The effect of methoxychlor on the epididymal antioxidant system of adult rats, Reprod. Toxicol. 16 (2) (2002) 161–172. [17] S. Vaithinathan, B. Saradha, P.P. Mathur, Transient inhibitory effect of methoxychlor on testicular steroidogenesis in rat: an in vivo study, Arch. Toxicol. 82 (11) (2008) 833–839.
[18] S. Vaithinathan, B. Saradha, P.P. Mathur, Methoxychlor-induced alteration in the levels of HSP70 and clusterin is accompanied with oxidative stress in adult rat testis, J. Biochem. Mol. Toxicol. 23 (1) (2009) 29–35. [19] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1) (1951) 265–275. [20] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (5259) (1970) 680–685. [21] I.P. Kapoor, R.L. Metcalf, R.F. Nystrom, G.K. Sangha, Comparative metabolism of methoxychlor, methiochlor, and DDT in mouse, insects, and in a model ecosystem, J, Agric. Food Chem. 18 (6) (1970) 1145–1152. [22] D.M. Stresser, D. Kupfer, Human cytochrome P450-catalyzed conversion of the proestrogenic pesticide methoxychlor into an estrogen: role of CYP2C19 and CYP1A2 in O-demethylation, Drug Metab. Dispos. 26 (9) (1998) 868– 874. [23] K. Williams, J. Frayne, E.A. McLaughlin, L. Hall, Expression of extracellular superoxide dismutase in the human male reproductive tract, detected using antisera raised against a recombinant protein, Mol, Hum. Reprod. 4 (3) (1998) 235–242. [24] J. Chandra, A. Samali, S. Orrenius, Triggering and modulation of apoptosis by oxidative stress, Free Radic. Biol. Med. 29 (3–4) (2000) 323–333. [25] Y.H. Ling, L. Liebes, Y. Zou, R. Perez-Soler, Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to Bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells, J. Biol. Chem. 278 (36) (2003) 33714–33723. [26] S. Nagata, Fas ligand-induced apoptosis, Annu. Rev. Genet. 33 (1999) 29–55. [27] M.O. Hengartner, The biochemistry of apoptosis, Nature 407 (6805) (2000) 770–776. [28] F. Romeo, D. Li, M. Shi, J.L. Mehta, Carvedilol prevents epinephrine-induced apoptosis in human coronary artery endothelial cells: modulation of Fas/Fas ligand and caspase-3 pathway, Cardiovasc. Res. 45 (3) (2000) 788–794. [29] J. Lee, J.H. Richburg, E.B. Shipp, M.L. Meistrich, K. Boekelheide, The Fas system, a regulator of testicular germ cell apoptosis, is differentially up-regulated in Sertoli cell versus germ cell injury of the testis, Endocrinology 140 (2) (1999) 852–858. [30] K Boekelheide, S.L. Fleming, K.J. Johnson, S.R. Patel, H.A. Schoenfeld, Role of Sertoli cells in injury-associated testicular germ cell apoptosis, Proc. Soc. Exp. Biol. Med. 225 (2) (2000) 105–115. [31] J. Lee, J.H. Richburg, S.C. Younkin, K. Boekelheide, The Fas system is a key regulator of germ cell apoptosis in the testis, Endocrinology 138 (5) (1997) 2081–2088. [32] S. Kreuz, D. Siegmund, J.J. Rumpf, D. Samel, M. Leverkus, O. Janssen, G. Hacker, O. Dittrich-Breiholz, M. Kracht, P. Scheurich, H. Wajant, NFkappaB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP, J. Cell. Biol. 166 (3) (2004) 369–380. [33] P. Kaur, G. Kaur, M.P. Bansal, Tertiary-butyl hydroperoxide induced oxidative stress and male reproductive activity in mice: role of transcription factor NFkappaB and testicular antioxidant enzymes, Reprod. Toxicol. 22 (3) (2006) 479–484. [34] M.D. Jacobson, M. Weil, M.C. Raff, Programmed cell death in animal development, Cell 88 (3) (1997) 347–354. [35] B. Saradha, S. Vaithinathan, P.P. Mathur, Lindane induces testicular apoptosis in adult Wistar rats through the involvement of Fas-FasL and mitochondriadependent pathways, Toxicology 255 (3) (2009) 131–139. [36] K. Shinoda, K. Mitsumori, K. Yasuhara, C. Uneyama, H. Onodera, M. Hirose, M. Uehara, Doxorubicin induces male germ cell apoptosis in rats, Arch. Toxicol. 73 (4–5) (1999) 274–281. [37] C. Strandgaard, M.G. Miller, Germ cell apoptosis in rat testis after administration of 1,3-dinitrobenzene, Reprod. Toxicol. 12 (2) (1998) 97–103. [38] J.B. Kerr, Spontaneous degeneration of germ cells in normal rat testis: assessment of cell types and frequency during the spermatogenic cycle, J. Reprod. Fertil. 95 (3) (1992) 825–830.
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Methoxychlor induces apoptosis via mitochondria- and FasL-mediated pathways in adult rat testis S. Vaithinathan, B. Saradha, P.P. Mathur ∗ Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Pondicherry 605 014, India
a r t i c l e
i n f o
Article history: Received 31 December 2009 Received in revised form 3 March 2010 Accepted 5 March 2010 Available online 12 March 2010 Keywords: Methoxychlor Testis Apoptosis Caspase NF-B FasL
a b s t r a c t In the past few years, there has been much concern about the adverse health effects of environmental contaminants in general and organochlorine in particular. Studies have shown the repro-toxic effects of long-term exposure to methoxychlor, a member of the organochlorine family. However, the insight into the mechanisms of gonadal toxicity induced by methoxychlor is not well known. In the present study we sought to elucidate the mechanism(s) underpinning the gonadal effects within hours of exposure to methoxychlor. Experimental rats were divided into six groups of four each. Animals were orally administered with a single dose of methoxychlor (50 mg/kg body weight) and killed at 0, 3, 6, 12, 24, and 72 h post-treatment. The levels and time-course of induction of apoptosis-related proteins like cytochorome C, caspase 3 and procaspase 9, Fas–FasL and NF-B were determined to assess sequential induction of apoptosis in the rat testis. DNA damage was assessed by TUNEL assay and flowcytometry. Administration of methoxychlor resulted in a significant increase in the levels of cytosolic cytochrome c and procaspase 9 as early as 6 h following exposure. Time-dependent elevations in the levels of Fas, FasL, pro- and cleaved caspase 3 were observed. The DNA damage was measured and showed time-dependent increase in the TUNEL positive cells, and also by flowcytometry of testicular cells. The study demonstrates induction of testicular apoptosis in adult rats following exposure to a single dose of methoxychlor. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In the recent years increased incidence of reproductive disorders has elevated concerns about the adverse effect of endocrinedisrupting chemicals on reproductive health in humans and wild life. Most of the endocrine disruptors released into the environment show estrogenic properties and exert their effects by mimicking endogenous hormones thereby leading to the disruption of endocrine mechanisms [1]. Methoxychlor is a broad spectrum chlorinated insecticide which is used to control insects on agricultural crops, livestock, animal feeds, barns, home, gardens and on household pets [2]. Thus, humans as well as wildlife species are exposed to methoxychlor easily. Methoxychlor acts as a proestrogen but it can be metabolized by the liver into mono-, bis-, tris-hydroxy and Catechol-M, which are estrogenic in nature than parental compound [3,4]. The most active estrogenic metabolite is bis-hydroxy methoxychlor(2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) [2,5]. Recent findings reveal that methoxychlor reduces fertility in male rats by reducing the weight of seminal vesicle, serum testosterone and dehydroepiandrosterone at a dose level of 200 mg/kg
∗ Corresponding author. Tel.: +91 413 2655212; fax: +91 413 2655211. E-mail address: [email protected] (P.P. Mathur). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.03.014
body weight for 7 days [6], ovarian atrophy and decreases the ability of ovarian cells to synthesize and secrete hormones in female rats [7,8]. The metabolite 2,2-bis-(p-hydroxyphenyl)-1,1,1trichloroethane (HPTE) is known to act as estrogen receptor ER ␣ agonist and ER  antagonist in human hepatoma cells [9]. Exposure to methoxychlor at doses of 0, 5, 50, and 150 mg/kg/day during the perinatal/juvenile period, reduced the spermatogenesis of males as adults by reducing their Sertoli cell number [10]. Oral exposure to methoxychlor caused atrophy of male sexual organs in rats at the dosage of 100 and 200 mg/kg/day for 70 days [11]. Methoxychlor exposure to pregnant female rats from embryonic days 8 to 15 at the dosage of 100 and 200 mg/kg/day showed an increase in spermatogenic cell apopotis and decreased sperm number and motility in adult animals of F1 and F2 generation [12,13]. Studies from our laboratory have exhibited a state of oxidative stress induced in adult rat testis following short- and longterm exposure of methoxychlor. Oxidative stress is induced by promoting lipid peroxidation, and a decrease in the activity of antioxidant enzymes in testis and epididymis [14–16]. Recently, we have demonstrated a transient inhibitory effect of methoxychlor (50 mg/kg body weight) on testicular steroidogenic enzymes, 5 3-hydroxysteroid dehydrogenase and 5 17-hydroxysteroid dehydrogenase within 6–12 h of exposure and a possible role of hydrogen peroxide (H2 O2 ) in mediating these effects was suggested [17]. Methoxychlor showed alteration in the levels of heat shock
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118 Table 1 Antibodies used in the present study. Vendor
Antibody
Host species
Catalog no.
Santa Cruz Biotechnology Santa Cruz Biotechnology Santa Cruz Biotechnology Santa Cruz Biotechnology Santa Cruz Biotechnology
Caspase 3 Caspase 9 Fas Fas-L NF-кB
Goat Rabbit Rabbit Rabbit Rabbit
SC-1225 SC-7885 SC-7886 SC-834 SC-7151
protein and clusterin accompanied by an induction of oxidative stress in the testis at a same dose [18]. Cell death by apoptosis is a part of normal development and maintenance of testicular homeostasis. During various stages of spermatogenesis, an adequate amount of germ cells are eliminated via the process of apoptosis in order to maintain a precise germ cell population in compliance with the supportive capacity of the Sertoli cells. The two divergent pathways of testicular apoptosis namely, the receptor-mediated Fas/FasL pathway and the mitochondrial-mediated pathway play an important role in maintaining the germ cell population [24]. Inappropriate activation of apoptosis could enormously jeopardize spermatogenesis and hence fertility. Reactive oxygen species (ROS) is considered a potential signal for apoptosis. Elevated levels of ROS can cause oxidation of the mitochondrial pores thereby disrupting the mitochondrial membrane potential and releasing cytochrome c thereby activating the mitochondrial-mediated pathway of apoptosis. In addition, ROS have been shown to induce the expression of Fas receptor and ligand stimulating the Fas/FasL-mediated apoptotic signal transduction pathway. Several environmental disruptors are known to inappropriately activate apoptosis in testicular locale by increasing the levels of ROS [29,35]. Taking a lead from these reports and our previous studies, we sought to investigate whether short-term treatment with a low dose of methoxychlor would alter the levels of apoptosis-related proteins in adult rat testis. The aim of the present study was to determine the effects of methoxychlor on testicular apoptosis and to investigate FasL-dependent pathway. We investigated the expressions of Fas, FasL, and activation of NF-B and to find whether caspase family members play an important role in apoptosis, it is also of interest to determine the expression levels of caspase 3 and procaspase 9 in methoxychlor-induced apoptosis.
111
galore, India) and water ad libitum. Seven days prior to the start of the experiments, rats were handled daily for 5 min to acclimatize them to human contact and minimize their physiological responses to handling. The experiments were carried out in accordance with the guidelines of the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA), Government of India (CPCSEA 2003) and were approved by the Institutional Animal Ethics Committee of Pondicherry University. Methoxychlor was dissolved in olive oil and administered (50 mg/kg body weight) to all treatment groups through oral gavage. Each group consisted of four animals. After treatment with methoxychlor for various times (0, 3, 6, 12, 24, and 72 h) animals were killed by cervical dislocation. Testes were collected, cleared of the adhering tissues, weighed and stored at −80 ◦ C until analysis.
2. Materials and methods 2.1. Chemicals Methoxychlor(1,1,1 trichloro-2,2-bis(4-methoxyphenyl) ethane, ∼95% pure) was purchased from Sigma Chemical Company (St. Louis, MO, USA). Details on primary antibodies used in this study are listed in Table 1. Goat anti-rabbit IgG and rabbit anti-goat IgG conjugated to FITC, goat anti-rabbit IgG conjugated to Cy3TM , horseradish peroxidase-conjugated goat anti-rabbit IgG and rabbit anti-goat IgG were obtained from Bangalore Genei (Bangalore, India). TUNEL kit was purchased from Promega, USA. All other chemicals used were of analytical grade and were obtained from local commercial sources. 2.2. Experimental design Adult male rats (80–90 days), of Wistar strain procured from an authorized rat vendor (M/S Raghavendra Enterprises, Bangalore, India), were housed in polypropylene cages and maintained at 22–25 ◦ C under a well-regulated light and dark (12:12 h) schedule in the animal house at Pondicherry University. The animals were provided with standard rat chow (Sai Durga Feeds and Foods, Ban-
Fig. 1. Effect of methoxychlor in the levels of cytochrome c, procaspase-9 and -3 in the testis of methoxychlor-treated rats over different time points. Methoxychlor (50 mg/kg) was administered to adult male rats (n = 4) which were killed at specific time points thereafter. (A) Immunoblots showing significant increase in the cytosolic levels of cytochrome c (11 kDa) from 6 h post-treatment. Immunoblot of procaspase 9 (37 kDa) shows a parallel increase from 6 h onwards. Maximum changes in the levels of cytochrome c and procaspse 9 are observed at 24 and 72 h post-treatment. The level of cytochrome c declines at 72 h, yet remains significantly higher than control. A sequential increase in the levels both procaspase 3 (32 kDa) and the cleaved fragment (p17) is observed from 6 to 72 h post-exposure. Immunoblot showing sequential increase in the levels of Fas (48 kDa) and FasL (30 kDa) from 6 to 24 h post-treatment. Immunoblot of NF-B shows time-dependent decrease in the cytosolic levels of NF-B p65 with a maximum decline at 12 h post-exposure. By 72 h, the levels of NF-B p65 in the cytosolic extract increase, yet remains significantly higher than control. Actin blot shows equal protein loading. Each of these depicted blots is representative of four experiments. (B) Graph representing densitometrically scanned results corresponding to the blots depicted in A. Each data point represents the average value from four independent experiments normalized against the control (0 h). The control was arbitrarily set at 1. Asterisk indicates values significantly different p < 0.05 as compared with control.
112
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
Fig. 2. Co-localized expression of caspase 3 and Fas in the testis of methoxychlor-treated rats; (a, d, g, and j) visualization of caspase 3 staining; (b, e, h, and k) visualization of Fas staining; (c, f, i, and l) visualization of overlap of Fas and caspase-3 staining. Fas and caspase 3 is localized to peritubular germ cells (spermatogonia and spermatocytes) and their coexistence is visualized (c, f, i, and l) when green (caspase 3) and red fluorescence (Fas) are superimposed (yellow to orange). A time-dependent increase in the expression of caspase 3 and Fas in the testis of methoxychlor-treated rats may be noted. The bar represents 50 m. (For interpretation of the references to color in this figure caption, the reader is referred to the web version of the article.)
2.3. Immunoblot analysis of caspase-3, -9, cytochrome c, Fas, FasL and NF-B Testes lysates were prepared in the lysis buffer (50 mM Tris, pH 7.4 containing 0.15 M NaCl, 10% glycerol (v/v), 1% NP-40 (v/v), 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 1 mM EDTA, 150 M bestatin, 1 M leupeptin and 1 M aprotinin) using a tissue:buffer ratio of 1:5. After homogenization, samples were first centrifuged at 1000 × g for 2 min to remove large tissue fragments and then centrifuged again at 10,000 × g for 30 min. The supernatants (cytoplasmic extract) were collected and stored at −70 ◦ C. The protein concentration was determined by Lowry’s method [19]
and equal quantities of protein were loaded per lane and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis [Mini Protean II System, Bio-Rad] as described by Laemmli [20]. Electrophoresis was performed at 75 V and the resolved proteins were electrophoretically transferred onto a nitrocellulose membrane (NYTRAN, Keene, NH, USA) in the transfer buffer (0.2 mol/L glycine, 25 mM Tris, and 20% methanol). Successful transfer was confirmed by Ponceau S staining of the blots. The membranes were incubated in a blocking buffer (phosphate-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dry milk powder) for 5 h at room temperature, followed by incubation in respective primary antibodies. Anti-caspase 3 was diluted at 1/100, and anti-
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
caspase 9, anti-cytochrome c, anti-Fas, anti-FasL and anti-NF-B were diluted at 1/200. The incubation with the primary antibodies was carried out overnight at 4 ◦ C. The following day, blots were washed in phosphate-buffered saline and incubated for 1 h at room temperature with either horseradish peroxidase-conjugated antirabbit IgG or anti-goat IgG (1:1000 dilution). Immunodetection of proteins was revealed by using tetramethylbenzidine/hydrogen peroxide (TMB/H2 O2 ) (Bangalore Genei, Bangalore, India) as a substrate and resulting immnunospecific bands were quantified by densitometry. 2.4. Immunofluorescent staining of Fas, FasL, caspase 3 and NF-B Rat testes were fixed according to Bouin’s method and embedded in paraffin and sectioned at a thickness of 5 m on poly-l-lysine-coated slides. After deparaffinization and rehydration, sections were washed in PBS (10 mM NaH2 PO4 , pH 7.4 at 22 ◦ C containing 0.15 M NaCl) and permeabilized in 0.2% Triton X100 (v/v). Sections were then blocked with 10% non-immune goat serum for 1 h. Sections were incubated with anti-FasL and anti-NFB (1:100 dilution each containing 1% non-immune goat serum) antibodies overnight at 4 ◦ C. After repeated rinsing with PBS, the sections were saturated with goat anti-rabbit IgG conjugated to FITC (1:100 dilution containing 10% non-immune goat serum) for 2 h in the dark. After being washed in PBS three to four times (5 min each), the slides were mounted and examined under the microscope (Olympus System, model CX 41RF, Japan). To determine whether Fas and caspase 3 were colocalized, the sections were sequentially incubated with appropriate combinations of antibodies. The tissue sections were incubated with primary antibody mixture, which consisted of rabbit anti-Fas and goat anti-caspase 3 (1:100 dilution each containing 1% nonimmune goat serum) overnight at 4 ◦ C. After repeated rinsing with PBS, the sections were incubated with secondary antibody mixture, which consisted of goat anti-rabbit IgG conjugated to Cy3TM and rabbit anti-goat IgG conjugated to FITC (1:100 dilution each containing 10% non-immune goat serum). After being washed in PBS three to four times (5 min each), the slides were mounted and examined under the microscope (Olympus System, model CX 41RF, Japan). 2.5. TUNEL assay Apoptotic cells were detected using the Dead End Colorimetric Apoptosis Detection System (Promega Corp., Madison, WI, USA). This system end-labels the fragmented DNA of apoptotic cells using a modified terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. It consists of incorporation of biotinylated nucleotides at the 3 -OH DNA ends using the terminal deoxynucleotidyl transferase, recombinant (rTDT) enzyme. Horseradishperoxidase-labeled streptavidin is then bound to these biotinylated nucleotides, which are detected using the peroxidase substrate, hydrogen peroxide and the stable chromogen diaminobenzidine (DAB). Briefly, the tissue sections were deparaffinized, rehydrated and fixed in 4% paraformaldehyde solution in PBS. Proteinase K (20 g/ml) treatment for 15 min was followed by a second fixation in 4% paraformaldehyde solution in PBS. The sections were incubated with equilibration buffer (200 mM potassium cacodylate, pH 6.6, 25 mM Tris–HCl, pH 6.6, 0.2 mM DTT, 0.25 mg/ml BSA, 2.5 mM cobalt chloride) for 10 min. Subsequently, the sections were allowed to react with rTDT reaction mixture (98 l equilibration buffer, 1 l biotinylated nucleotide mix and 1 l rTDT enzyme) for 1 h at 37 ◦ C in a humidified chamber. The slides were immersed in 2× SCC for 15 min to stop the reaction and the endogenous peroxidase were blocked by incubating the sections with 0.3% H2 O2 in PBS for 5 min. After
113
incubation with streptavidin–HRP solution for 30 min at room temperature, the slides were stained with DAB–H2 O2 in the dark. Positive nuclei stained dark-brown and were visualized under a light microscope. Negative controls with TUNEL assay were performed according to the instructions provided by the manufacturer. 2.6. Flowcytometry analysis Testicular suspensions were prepared by the following method. Briefly, the tunica albuginea was removed and the decapsulated testis was placed in ice cold nutrient medium (Dulbecco’s modified eagle medium) and minced gently with razor. Then the tissue was transferred to nutrient medium containing 0.25% collagenase (Type IA) (Sigma, St. Louis, USA) for 30 min in a shaking water bath at 32.5 ◦ C. The cell suspension was filtered through a 100-m and 40-m nylon mesh (BD Falcon, Bedford, USA) in order to discard tissue debris and to obtain single cell suspension, respectively. The suspension was centrifuged at 300 × g for 5 min and the pellet was washed twice with medium. One aliquot of each cell suspension was stained with 0.2% trypan blue for the determination of viability. The cells were fixed in ice cold 70% methanol and stored at 20 ◦ C for minimum 24 h. After fixation cells were incubated with 0.25% RNase (Bangalore Genei, India) (37 ◦ C, 20 min). After washing twice with PBS, DNA staining was carried out by Propidium Iodide (PI; Sigma, St. Louis, USA) solution (50 g/ml in PBS), and placed in the dark for 30 min at 4 ◦ C. Enumerations of cells on the basis of their DNA content were carried out in a Flow Cytometer (FACS Calibur, Becton-Dickinson, USA) equipped with air-cooled 15 mv Argon ion laser at 488 nm and the emitted light from cells was detected at 580 ± 30 nm on FL2. The DNA histograms were analyzed by Cell Quest (Becton-Dickinson, USA) software. 2.7. Statistical analysis Data were expressed as mean ± SD for four animals per group. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s post-test using SPSS (student version 7.5, SPSS Inc., UK). p < 0.05 was taken as statistically significant. Images were compiled using Adobe Photoshop (version 7.0, San Jose, CA). Densitometric scanning was performed using Gene tool (version 3.05, Synoptics Ltd., Cambridge, UK). 3. Results 3.1. Expression of cytochrome c, caspase 9 and caspase 3 proteins in methoxychlor-treated rats To assess the effect of methoxychlor on the mitochondriadependent cell death pathway, the levels of cytosolic cytochrome c, procaspase 9 and caspase 3 proteins were evaluated in adult rat testis. Western blot analysis revealed a significant elevation in the levels of cytosolic cytochrome c (11 kDa) and procaspase 9 (37 kDa) as early as 6 h following exposure to a single dose of methoxychlor (Fig. 1). Maximal changes in the levels of cytochrome c and procaspse 9 were observed at 12 and 72 h post-treatment. A time-dependent elevation in the levels of procaspase 3 (32 kDa) accompanied by an increase in the cleaved fragment (17 kDa) was observed following exposure to a single dose of methoxychlor (Fig. 1). 3.2. Effect of methoxychlor on the protein levels of Fas, FasL and NF-B Cytoplasmic extract of the testis was used to assess the effect of methoxychlor on Fas–FasL system by western blot analysis
114
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
Fig. 3. Immunofluorescent staining of FasL in the testis of methoxychlor-treated rats. Representative images for FasL immunoreactivity in control (a), 6 h (b), 12 h (d) and 72 h (f) post-treated groups. (c, e, and g) Magnified images of (b, d, and f), respectively. FasL is localized to the cytoplasmic extensions of Sertoli cells (arrows heads) and peritubular germ cells (asterisks). A time-dependent increase in the expression of FasL in the testis of rats exposed to a single dose of methoxychlor may be noted. The bar represents 50 m.
and to delineate the possible role of NF-B-mediated effects. A significant increase in the levels of Fas and FasL in a timedependent manner from 6 h onwards reaching a peak at 24 h in the methoxychlor-treated rats was observed (Fig. 1). On the contrary, a time-dependent decrease in the levels NF-B, with a significant decline at 12 and 24 h was resulted. However, the levels started recouping at 72 h post-treatment (Fig. 1). 3.3. Colocalized expression of caspase 3 and Fas Co-localization of Fas and Caspase 3 showed a time-dependent increase in the methoxychlor-treated rats. Fas and caspase 3 were localized in spermatogonia and spermatocytes during double immunofluorescent labeling, and their coexistence was visualized
Fig. 4. Immunofluorescent staining of NF-B p65 in the testis of methoxychlortreated rats. Representative images for NF-B p65 immunoreactivity in control (a), 6 h (c), 24 h (e) and 72 h (g) post-treated groups. (b, d, f, and h) Magnified image of (a, c, e, and g) respectively. A time-dependent increase in nuclear localization of NF-B p65 in spermatogonia (asterisks) and spermatocytes (arrow heads) with a maximal nuclear staining at 24 h post-exposure (e and f) may be noted. At 72 h, the nuclear staining is less pronounced (g and h). The bar represents 50 m.
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
115
Fig. 5. Methoxychlor-induced germ cell death in the testis of rats as revealed by TUNEL assay. Representative images for TUNEL staining in negative control (a), control (b), 6 h (c), 12 h (d) and 72 h (e) post-treatment groups. The TUNEL-positive cells (arrows) are found in the peripheral region near the basement membrane of the seminiferous tubules. A time-dependent increase in the number of apoptotic cells in the testis of methoxychlor-treated rats may be noted. The bar represents 50 m.
when green and red fluorescence were superposed (yellow to orange in Fig. 2).
labeled germ cells increased progressively. TUNEL-labeled germ cells were principally spermatogonia and spermatocytes in the methoxychlor-treated rats.
3.4. Immunolocalization of FasL and NF-B in testis of methoxychlor-treated rats
3.6. Flowcytometry analysis of apoptosis
Expression of FasL and NF-B protein in the methoxychlor exposed rat testis was examined by immunofluorescent microscopy. Cytoplasm of Sertoli cells and peritubular cells showed a FasL expression. In a time-dependent manner from 6 to 72 h immunoreactivity of FasL showed an increase in the testis (Fig. 3). In methoxychlor-treated rats NF-B p65 subunit changed from cytoplasm to nucleus of germ cells. This change in the localization of NF-B p65 subunit was observed from 3 h with maximal nuclear reactivity at 12 h post-expsoure (Fig. 4). However, at 72 h the nuclear localization of p65 subunit was less pronounced and only a few tubules displayed nuclear reactivity in germ cells.
Total germ cell number was determined using flowcytometric analysis of propidium iodide labeled cells (Fig. 6). We detected three major histogram peaks of DNA content, which represented M1 (elongating and elongated spermatid), haploid-M2 (round spermatid), diploid-M3 (spermatogonia, preleptotene primary and secondary spermatocytes) and tetraploid-M4 (spermatogonia, leptotene). Compared with control animals, 6 h post-treatment of methoxychlor showed a time-dependent increase in elongated cells (Figs. 6 and 7). A time-dependent decrease of diploid cells occurs in post-treatment of methoxychlor. These results correlate with the TUNEL results.
3.5. TUNEL assay
4. Discussion
In situ TUNEL analysis quantitative data from TUNEL-labeled germ cells are shown in Fig. 6. In the testes when compared to the control rats (3.25 ± 1.02), the percentage of TUNEL-labeled germ cells after a single dose of methoxychlor increased in the peripheral regions in time-dependent manner at 6 h (8.66 ± 2.10) and 72 h (21.98 ± 3.21) post-treatment (Fig. 5). The number of TUNEL-
The current study was conducted to interpret the testicular response to methoxychlor in adult male rats. Literature reveals metabolism and biotransformation of methoxychlor in rats and its elimination by the system within 48 h following the oral exposure [21,22]. Our previous studies have demonstrated the induction of oxidative stress and inhibition of steroidogenesis to a single expo-
116
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
sure of methoxychlor at 50 mg/kg body weight [17,18]. Knowledge on this terrain as well as the high vulnerability of the testis to methoxychlor prompted us to study the stress-mediated apoptosis following exposure to single dose of methoxychlor. The results of the present study indicate that methoxychlor, at a concentration of 50 mg/kg body weight, has the ability to induce apoptosis in testicular cells in vivo after 12 h of exposure. The present study sought to investigate the sequential changes in the levels of cytochrome c, procaspase 9 and caspase 3, Fas and FasL proteins in the testis of adult rats following exposure to a single dose of methoxychlor. There is a universal consent that male reproductive organs are particularly susceptible to the detrimental effects of reactive oxygen species (ROS) and lipid peroxidation, which lead to impaired fertility [23]. Reactive oxygen species (ROS) is considered a potential signal for apoptosis. There is evidence that ROS plays a role in the induction of both death receptor and mitochondrial-apoptotic pathways [24]. Particularly in the testes, the germ cells such as spermatozoa are highly receptive to ROS damage and damage from ROS-generated products due to the prevalence of oxidation-prone unsaturated fatty acids on their plasma membrane. Apoptosis involves a cascade of signals and there are different pathways involved in testicular cell apoptosis pertaining to specific treatments. Literature reveals as a consequence of ROS generation in cells, mitochondrial dysfunction should occur [25]. In the present study methoxychlor treatment showed a significant increase in the level of cytoplasmic cytochrome c at 6–24 h following exposure. A significant elevation of procaspase 9 was observed in 6 h of posttreatment. The changes in the levels of both proteins coincide at the same time points. The results indicate that cytochrome c acti-
vates procaspase 9, which in turn activates executioner caspases, leading to the intrinsic pathway of apoptosis. The maximal levels of cytochrome c and procaspase 9 coincide with our previous studies where the decrease in the antioxidant enzymes levels and accumulation of lipid peroxidation [18]. In this study, we observed a significant increase in the activation of caspase 3 followed by an elevation in cleaved caspase 3 from 6 h of post-treatment. Elevation in the level of cleaved caspase 3 after the release of cytochrome c accompanied by an increase in procaspase 9 suggest the activation of intrinsic pathway of apoptosis in the testis of treated rats. Methoxychlor-induced apoptosis in the testis may be caused by increased oxidative stress after the treatment of methoxychlor. The Fas system has been widely recognized as a key regulator of the apoptosis signal transduction pathways. Fas is a type I transmembrane receptor protein and Fas ligand (FasL) is a type II transmembrane protein. The ligation of FasL to Fas triggers an FasLmediated apoptotic death in a target cell leading to the activation of caspase 3 [26,27]. All the cells expressing both Fas and FasL do not undergo apoptosis under normal conditions, but when an external stimuli is induced then can become significantly sensitized to the Fas-mediated apoptosis [28]. Our findings revealed an evident induction of both Fas and FasL levels in testis after methoxychlor treatment, when in comparison to control groups. The highest expression of FasL protein was detected at 24 h post-treatment of methoxychlor, while the levels of Fas protein reached to the maximum at 24 h when the level of apoptosis also peaked. This result was in accordance to onset of FasL mRNA upregulation after Sertoli cell injury [29]. Immunofluorescent staining showed a timedependent expression of FasL in the Sertoli cells and spermatogonia
Fig. 6. Flow cytometric analysis of testicular cells from control and methoxychlor-treated rats. Four different cell populations corresponding to four different peaks such as elongating and elongated spermatid as M1, haploid cells as M2 (round spermatid), diploid cells like spermatogonia, preleptotene primary and secondary spermatocytes as M3 and tetraploid cells, spermatogonia, leptotene as M4 are observed in control group. There was a decrease in time-dependent manner in the amplitude of haploid cells in the methoxychlor-treated groups.
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
117
Fig. 7. Germ cells isolated from testes of control and methoxychlor-treated rats (live versus dead-arrow). Number of viable cells reduced time-dependent manner in methoxychlor-treated rats (a–d).
at 6–72 h post-treatment of methoxychlor. The result indicated that increased expression of FasL in Sertoli cells triggers apoptotic elimination of Fas-expressing germ cells, suggests that the upregulation of FasL expression in Sertoli cells and increased activation of caspase 3 may be associated with apoptosis in methoxychlor-treated rats. Co-localization of caspase 3 and Fas showed an increased expression of caspase 3 and Fas at 12–72 h of post-treatment which correlates with our earlier result acquired by western blot. This shows that both the pathways occur in same cell type in the methoxychlor-treated rats when compared to control animals. The results further confirm the involvement of Fas-mediated pathway in methoxychlor-induced apoptosis. Animals when exposed to specific testicular toxicants, such as 2,5-hexanedione or mono2-ethylhexyl phthalate, which target the germ cell and Sertoli cells and disrupt their functions, will lead to up-regulation of Fas and FasL in the injured cells, respectively. This up-regulation result in a Fas–FasL-mediated elevation of germ cell apoptosis [29–31]. The ligation of Fas to FasL could stimulate NF-B activation [32]. NF-B is a complex of p65 and p50 proteins and is found to be constitutively expressed in the nuclei of rat testicular cells. In normal cells, NF-B dimers are sequestered in the cytoplasm by inhibitory kappa B (IB). When it exposed to external signal degradation of IkB, free NF-B translocates to the nucleus. NF-B exerts both proand antiapoptotic effects in testis upon toxicant exposure [33]. Our study demonstrated that single dose exposure to methoxychlor could decrease the level of p65 subunit of NF-B in cytoplasmic homogenate of the testis at 6 h of post-treatment. The maximum reduction of NF-B occurs at 12 h and started to return normal at 72 h of post-treatment. Immunofluoresence results showed that nuclear translocation of NF-B in germ cells of methoxychlortreated rats from 6 h with a maximum of 24 h of post-treatment
which shows an apoptotic role of NF-B in testis. Both intrinsic and extrinsic pathways of apoptosis enter into a common downstream effector, caspase 3, which can induce DNA fragmentation by the activation of endonuclease. In this study, methoxychlor-induced apoptosis in male rat germ cells in a time-dependent manner. TUNEL-labeled germ cells induced by methoxychlor reached a maximum in number at 12 h after dosing. Cell death by apoptosis is a relatively rapid mechanism, and complete removal of the cell often occurs within hours from the tissue [34]. This has been confirmed by previous reports [35–37]. Thus, germ cell cytotoxicity exerts within a short period of methoxychlor treatment. Germ cell apoptosis in the rodent testes has also been observed as a part of normal spermatogenesis [38], which is increased by testicular toxicants. In addition, support to this TUNEL observation came from the fact that there was a time-dependent reduction of haploid cells as observed by flowcytometry. In conclusion, exposure to a single dose of methoxychlor induces apoptosis via mitochondria-mediated and FasL-dependent pathways in testis of adult rats. Conflict of interest None. Acknowledgements P.P. Mathur acknowledges the receipt of financial support from the Department of Science and Technology, Government of India under the projects (1) SP/SO/B-65/99 (2) DST-FIST and (3) Indian Council of Medical Research, New Delhi. B. Saradha acknowledges the Indian Council of Medical Research, New Delhi, India for Senior Research Fellowship. The authors also thank the staff of Bioinfor-
118
S. Vaithinathan et al. / Chemico-Biological Interactions 185 (2010) 110–118
matics Center, Pondicherry University, Pondicherry for providing various facilities. References [1] B. Saradha, P.P. Mathur, Effect of environmental contaminants on male reproduction, Environ. Toxicol. Pharmacol. 21 (1) (2006) 34–41. [2] A.M. Cummings, Methoxychlor as a model for environmental estrogens, Crit. Rev. Toxicol. 27 (4) (1997) 367–379. [3] Y. Hu, D. Kupfer, Enantioselective metabolism of the endocrine disruptor pesticide methoxychlor by human cytochromes P450 (P450 s): major differences in selective enantiomer formation by various P450 isoforms, Drug Metab. Dispos. 30 (12) (2002) 1329–1336. [4] Y. Hu, D. Kupfer, Metabolism of the endocrine disruptor pesticidemethoxychlor by human P450 s: pathways involving a novel catechol metabolite, Drug Metab. Dispos. 30 (9) (2002) 1035–1042. [5] S.S. Dehal, D. Kupfer, Metabolism of the proestrogenic pesticide methoxychlor by hepatic P450 monooxygenases in rats and humans: dual pathways involving novel ortho ring-hydroxylation by CYP2B, Drug Metab. Dispos. 22 (6) (1994) 937–946. [6] E.P. Murono, R.C. Derk, Y. Akgul, In vivo exposure of young adult male rats to methoxychlor reduces serum testosterone levels and ex vivo Leydig cell testosterone formation and cholesterol side-chain cleavage activity, Reprod. Toxicol. 21 (2) (2006) 148–153. [7] E.M. Martinez, W.J. Swartz, Effects of methoxychlor on the reproductive system of the adult female mouse: 2. Ultrastructural observations, Reprod. Toxicol. 6 (1) (1992) 93–98. [8] W.J. Swartz, M. Corkern, Effects of methoxychlor treatment of pregnant mice on female offspring of the treated and subsequent pregnancies, Reprod. Toxicol. 6 (5) (1992) 431–437. [9] K.W. Gaido, S.C. Maness, D.P. McDonnell, S.S. Dehal, D. Kupfer, S. Safe, Interaction of methoxychlor and related compounds with estrogen receptor alpha and beta, and androgen receptor: structure-activity studies, Mol, Pharmacology 58 (4) (2000) 852–858. [10] R.E. Chapin, M.W. Harris, B.J. Davis, S.M. Ward, R.E. Wilson, M.A. Mauney, A.C. Lockhart, R.J. Smialowicz, V.C. Moser, L.T. Burka, B.J. Collins, The effects of perinatal/juvenile methoxychlor exposure on adult rat nervous, immune, and reproductive system function, Fundam, Appl. Toxicol. 40 (1) (1997) 138–157. [11] H.S. Bal, Effect of methoxychlor on reproductive systems of the rat, Proc. Soc. Exp. Biol. Med. 176 (2) (1984) 187–196. [12] M.D. Anway, A.S. Cupp, M. Uzumcu, M.K. Skinner, Epigenetic transgenerational actions of endocrine disruptors and male fertility, Science 308 (5727) (2005) 1466–1469. [13] A.S. Cupp, M. Uzumcu, H. Suzuki, K. Dirks, B. Phillips, M.K. Skinner, Effect of transient embryonic in vivo exposure to the endocrine disruptor methoxychlor on embryonic and postnatal testis development, J. Androl. 24 (5) (2003) 736–745. [14] C. Latchoumycandane, P.P. Mathur, Induction of oxidative stress in the rat testis after short-term exposure to the organochlorine pesticide methoxychlor, Arch. Toxicol. 76 (12) (2002) 692–698. [15] C. Latchoumycandane, P.P. Mathur, Effect of methoxychlor on the antioxidant system in mitochondrial and microsome-rich fractions of rat testis, Toxicology 176 (1–2) (2002) 67–75. [16] C. Latchoumycandane, K.C. Chitra, P.P. Mathur, The effect of methoxychlor on the epididymal antioxidant system of adult rats, Reprod. Toxicol. 16 (2) (2002) 161–172. [17] S. Vaithinathan, B. Saradha, P.P. Mathur, Transient inhibitory effect of methoxychlor on testicular steroidogenesis in rat: an in vivo study, Arch. Toxicol. 82 (11) (2008) 833–839.
[18] S. Vaithinathan, B. Saradha, P.P. Mathur, Methoxychlor-induced alteration in the levels of HSP70 and clusterin is accompanied with oxidative stress in adult rat testis, J. Biochem. Mol. Toxicol. 23 (1) (2009) 29–35. [19] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1) (1951) 265–275. [20] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (5259) (1970) 680–685. [21] I.P. Kapoor, R.L. Metcalf, R.F. Nystrom, G.K. Sangha, Comparative metabolism of methoxychlor, methiochlor, and DDT in mouse, insects, and in a model ecosystem, J, Agric. Food Chem. 18 (6) (1970) 1145–1152. [22] D.M. Stresser, D. Kupfer, Human cytochrome P450-catalyzed conversion of the proestrogenic pesticide methoxychlor into an estrogen: role of CYP2C19 and CYP1A2 in O-demethylation, Drug Metab. Dispos. 26 (9) (1998) 868– 874. [23] K. Williams, J. Frayne, E.A. McLaughlin, L. Hall, Expression of extracellular superoxide dismutase in the human male reproductive tract, detected using antisera raised against a recombinant protein, Mol, Hum. Reprod. 4 (3) (1998) 235–242. [24] J. Chandra, A. Samali, S. Orrenius, Triggering and modulation of apoptosis by oxidative stress, Free Radic. Biol. Med. 29 (3–4) (2000) 323–333. [25] Y.H. Ling, L. Liebes, Y. Zou, R. Perez-Soler, Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to Bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells, J. Biol. Chem. 278 (36) (2003) 33714–33723. [26] S. Nagata, Fas ligand-induced apoptosis, Annu. Rev. Genet. 33 (1999) 29–55. [27] M.O. Hengartner, The biochemistry of apoptosis, Nature 407 (6805) (2000) 770–776. [28] F. Romeo, D. Li, M. Shi, J.L. Mehta, Carvedilol prevents epinephrine-induced apoptosis in human coronary artery endothelial cells: modulation of Fas/Fas ligand and caspase-3 pathway, Cardiovasc. Res. 45 (3) (2000) 788–794. [29] J. Lee, J.H. Richburg, E.B. Shipp, M.L. Meistrich, K. Boekelheide, The Fas system, a regulator of testicular germ cell apoptosis, is differentially up-regulated in Sertoli cell versus germ cell injury of the testis, Endocrinology 140 (2) (1999) 852–858. [30] K Boekelheide, S.L. Fleming, K.J. Johnson, S.R. Patel, H.A. Schoenfeld, Role of Sertoli cells in injury-associated testicular germ cell apoptosis, Proc. Soc. Exp. Biol. Med. 225 (2) (2000) 105–115. [31] J. Lee, J.H. Richburg, S.C. Younkin, K. Boekelheide, The Fas system is a key regulator of germ cell apoptosis in the testis, Endocrinology 138 (5) (1997) 2081–2088. [32] S. Kreuz, D. Siegmund, J.J. Rumpf, D. Samel, M. Leverkus, O. Janssen, G. Hacker, O. Dittrich-Breiholz, M. Kracht, P. Scheurich, H. Wajant, NFkappaB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP, J. Cell. Biol. 166 (3) (2004) 369–380. [33] P. Kaur, G. Kaur, M.P. Bansal, Tertiary-butyl hydroperoxide induced oxidative stress and male reproductive activity in mice: role of transcription factor NFkappaB and testicular antioxidant enzymes, Reprod. Toxicol. 22 (3) (2006) 479–484. [34] M.D. Jacobson, M. Weil, M.C. Raff, Programmed cell death in animal development, Cell 88 (3) (1997) 347–354. [35] B. Saradha, S. Vaithinathan, P.P. Mathur, Lindane induces testicular apoptosis in adult Wistar rats through the involvement of Fas-FasL and mitochondriadependent pathways, Toxicology 255 (3) (2009) 131–139. [36] K. Shinoda, K. Mitsumori, K. Yasuhara, C. Uneyama, H. Onodera, M. Hirose, M. Uehara, Doxorubicin induces male germ cell apoptosis in rats, Arch. Toxicol. 73 (4–5) (1999) 274–281. [37] C. Strandgaard, M.G. Miller, Germ cell apoptosis in rat testis after administration of 1,3-dinitrobenzene, Reprod. Toxicol. 12 (2) (1998) 97–103. [38] J.B. Kerr, Spontaneous degeneration of germ cells in normal rat testis: assessment of cell types and frequency during the spermatogenic cycle, J. Reprod. Fertil. 95 (3) (1992) 825–830.