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. 2017 Jan 6;6(2):223–231. doi: 10.1039/c6tx00315j

Endosulfan induces apoptosis by activating the negative regulation pathway of cell cycle and death receptor pathway in spermatogenic cells

Fang-Zi Guo a,b,†, Ying Xu a,b,†, Li-Hua Ren a,b,†, Jin Zhang a,b, Feng Zhang c, Junchao Duan a,b, Xian-Qing Zhou a,b,✉, Zhi-Wei Sun a,b
PMCID: PMC6060701  PMID: 30090493

graphic file with name c6tx00315j-ga.jpgThe male reproductive toxicity of endosulfan has been proved.

Abstract

The male reproductive toxicity of endosulfan has been proved. Nevertheless, the underlying molecular mechanisms of the apoptosis caused by endosulfan in spermatogenic cells remains poorly understood. In order to investigate the reproductive toxicity mechanism caused by endosulfan, there were four groups, which had eight Wistar male rats randomly assigned to them, and the rats in different groups received different doses of endosulfan for a period of 21 days. GC-1 spermatogenic cell lines were divided into four groups, and each group was exposed to different doses of endosulfan for 24 hours. The results of this research showed that endosulfan decreased the cell viability, damaged cell membranes and induced apoptosis in spermatogenic cells. Endosulfan had obviously activated the protein expression of PKC-δ, p53, p21cip1, p27kip1, Fas, FasL, Caspase-8, Caspase-3, and inhibited the expression of E2F-1. Endosulfan also induced oxidative stress and DNA damage in spermatogenic cells. The results of this research suggested that endosulfan could lead to E2F-1-induced apoptosis of spermatogenic cells by activating the negative regulation factors of the cell cycle, and endosulfan might cause apoptosis by death receptor pathway, causing oxidative stress.

Introduction

Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepine-3-oxide, C9H6Cl6O3S), a persistent organic pollutant (POP), is a widely used agricultural pesticide in developing countries.1,2 Massive amounts of endosulfan still exist in the environment.2 Some experts have detected endosulfan and its degradation products (endosulfan sulfate) in soy production regions.3 Endosulfan with a half-life of more than 14 d exists in water. Meanwhile, endosulfan with a longer half-life of two months to over two years persists in soil.4 The endosulfan pollution has spread in the air, water and soil environments.

Endosulfan is highly lipophilic and this can lead to bioaccumulation and biomagnification, resulting in adverse effects on organisms. Earlier studies have shown that endosulfan could affect non-target organisms such as fish, Daphnia and other insects which are not pests and also higher mammals including humans.5,6 Endosulfan can cause cardiovascular toxicity,7,8 neurotoxicity,9 hepatotoxicity,10 immunotoxicity11 and reproductive toxicity.12,13 There has been concern and more and more attention has been given to the reduction of biodiversity resulting from damaging the male reproductive system.14

It was shown in a previous study that endosulfan was a cause of male reproductive toxicity. Testicular sperm count and sperm motility were decreased and the sperm abnormality rate was induced and increased by endosulfan in Microtus oeconomus.15 This previous study also showed that endosulfan could inhibit spermatogenesis, damage the integrity of sperm chromatin, the histological structures and ultrastructures of testicles, and decrease the level of serum testosterone.16,17 Additionally, endosulfan led to the catabolism of spermatogenic cells in mice.18 A previous study by Wang et al. indicated that endosulfan could damage the mitochondrial structure, leading to dysfunction of energy metabolism resulting from oxidative stress.16 Takhshid et al. also proved that endosulfan caused reproductive toxicity by creating oxidative stress.19 However, the mechanism by which endosulfan leads to apoptosis resulting from oxidative stress in spermatogenic cells has so far remained unknown. Thus, the experiment reported in this paper was designed to investigate the role of endosulfan on the negative regulation pathway of the cell cycle and the death receptor pathway, in order to explore the possible toxic molecular mechanism of endosulfan and how to prevent it.

Materials and methods

Animals and treatments in vivo

Twelve week old, pathogen free, male Wistar rats with a weight of 280–320 g were raised in a standard cage (15 cm × 15 cm × 26 cm), and were obtained from the Weitong-Lihua Experimental Animal Center (Beijing, China; the license number of animal production was SCXK 2012-0001). The rats lived in standard laboratory conditions on a day/night cycle (12 h : 12 h) at the temperature of 20–24 °C and were supplied with standard food pellets and unlimited drinking water. The experiments were carried out under the institutional guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health after a one week adaption period. The design was permitted by the Committee on the Ethics of Animal Experiments of the Capital Medical University, Beijing, China.

Endosulfan, which was provided by Jiangsu Kuaida Agrochemical Co, Ltd, (Jiangsu, China) was administered via oral gavage by dissolving it in corn oil. Rats were randomly and averagely divided into four groups, with eight rats per group. Endosulfan was given in different doses (1, 5 or 10 mg per day per weight) to the experimental groups daily for a period of 21 days. The lowest dose of endosulfan was similar to the levels of endosulfan found in the environment,20 and the highest dose of endosulfan used was equivalent to the LD50 levels of endosulfan for rats given it by gavage,21 and the moderate dose of endosulfan in this experiment was the dose between the highest and the lowest. Rats in the control group were only given the isopycnic corn oil. The individual body weight on every second day was used to correct the exact dosage of the endosulfan for each rat. After the 21 successive treatments, the animals were weighed and then using 5 ml kg–1 of chloral hydrate (7%) by intraperitoneal injection, the animals were sacrificed. Next, the epididymides were collected quickly for analysis. Then the testes were removed, weighed, and stored at –20 °C before the next experiments.

Cell culture and treatment in vitro

The GC-1 spg line, was obtained from the American Type Culture Collection. The spermatogenic cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, USA) with 10% fetal bovine serum (Gibco, USA), 100 U ml–1 penicillin and 100 μg ml–1 streptomycin at 37 °C in a 5% carbon dioxide (CO2) humidified environment. Endosulfan was dissolved in dimethylsulfoxide (DMSO) which was then added to the culture solution. Because the final DMSO concentration in the culture solution was 0.36% (v/v), the viability of the GC-1 spg cells were not impacted by the presence of the DMSO. The spermatogenic cells in the control group were supplied only with isopycnic DMEM. Cells were cultured for 24 hours and then endosulfan was added to culture solution at concentrations of 0, 6, 12 and 24 μg ml–1 for another 24 h. Three replicate wells were used in each treatment group. All the experiments were repeated no less than three times.

Determination of apoptosis level

The terminal deoxyribonucleotide transferase-mediated nick-end labeling assay (TUNEL) kit (KeyGEN Biotech Co., Ltd, Nanjing, China) was used to detect apoptosis cells in sections of testis. Briefly, after dewaxing and hydration of the testis sections, they were washed three times with phosphate buffered saline (PBS), and then 1% Triton X-100 was used for 3 min and 3% hydrogen peroxide solution was added for 10 min. After that the sections were washed again with PBS, and then the sections were covered with protease K at 37 °C for 30 min. The addition of biotin-conjugated 2′-deoxyuridine 5′-triphosphate (dUTP) to the 3′-OH ends of the DNA fragments was catalyzed using terminal deoxynucleotidyl transferase (TdT). After rinsing with PBS, the sections were covered with streptavidin–horseradish peroxide solution at 37 °C for 30 min, then were placed in 3,3′-diaminobenzidine (DAB) and then stained with hematoxylin solution, Harris modified. The computer-aided software was used to analyse 20 different fields under a X71-F22PH light microscope (Olympus, Japan). The apoptosis of GC-1 spg was determined using the cell apoptosis assay (KeyGEN Biotech Co., Ltd, Nanjing, China). The assay was used according to the instructions, and the apoptosis cells were finally detected using flow cytometry (Becton–Dickinson, USA).

Measurement of cell viability

In order to detect the cell activity, a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT assay) was used. The working solution was added to cover the cells at 37 °C with 5% CO2 for 4 h and after that the cells were cultured with endosulfan for 24 h. Then 100 μL of DMSO was added at 37 °C for 30 min. Finally, a microplate reader (BIO-RAD, Japan) was used to detect the absorbance at 550 nm.

Measurement of the activities of lactic acid dehydrogenase (LDH)

The supernatants which were obtained at the end of the cell culture with endosulfan for 24 hours were used for the detection of the activity of LDH. The assay kit was used according to the instructions given and the activities of LDH were determined at 450 nm using a microplate reader (Bio-Rad, Japan).

Determination of oxidative stress

In order to detect malondialdehyde (MDA) which is the product of lipid peroxidation, the MDA kit (Jiancheng, China) was used. First of all, the testis was weighed, added to 100 mmol L–1 phosphate buffer and ground in an ice-water bath. Then after centrifuging the mixed liquid at 1000 rpm for 10 min, the supernatant was analyzed to determine the MDA content. The supernatant protein concentration was determined using the bicinchoninic acid (BCA) protein assay (Dingguo Changsheng Biotechnology Co. Ltd, China). Reactive oxygen species (ROS) were determined using the ROS kit (Jiancheng, China) according to the instructions given. Finally, the absorbance of the samples was read at 525 nm using a flow cytometry instrument (Becton–Dickinson, USA).

Immunocytochemistry detection of 8-hydroxy-2′-deoxyguanosine (8-OHdG), Fas and FasL

A modification of the method from Tsai et al. (2011)22 was employed. Briefly, GC-1 spg cells were incubated with 0, 6, 12 or 24 μg ml–1 endosulfan for 24 h on a coverslip. Then the cells were washed twice with PBS. Then 4% cold paraformaldehyde was used to fix the sections. DNA was denatured for 5 min at 4 °C and then treated with 0.1% Triton X-100 for 5 min at 4 °C and 10% normal goat serum used to block. Subsequently, the primary antibody was incubated with the sections at 4 °C overnight. The biotin-conjugated second antibody was then incubated with the sections for 10 min and then the streptavidin–peroxidase was added for another 10 minutes. Finally, the color was revealed using diaminobenzidene. A BX-51 camera (Olympus, Omachi, Japan) fitted to a microscope was used to take pictures.

Measurement of Caspase-3 and Caspase-8 activities

In order to assess the activities of Caspase-3 and Caspase-8 of GC-1 spg cells, Caspase assays were performed (Biovision, USA). After the cells were lysed using lysis buffer, the buffer containing the cells was centrifuged at 10 000g for 10 min. Then the supernatant which was added to the reaction buffer and the mixture was incubated at 37 °C for 1.5 h. At the end, a microtiter plate reader (Bio-Rad, Japan) was used for detection at 405 nm.

Determinations of protein expressions for Fas/FasL and regulatory factors of the cell cycle

A protein extraction kit (KeyGen, China) and the BCA protein assay (Dingguo Changsheng Biotechnology Co. Ltd, China) were used to extract and measure the total protein of the testis tissue. Western blotting was performed to assess the protein levels of Fas, FasL, PKC-δ, p53, p21cip1, p27kip1 and E2F-1. The analysis was performed using 12% separation gels, 5% spacer gel and poly(vinylidene fluoride) (PVDF) membranes (Millipore, USA). The blocking agent was 5% non-fat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST). The membrane was incubated with PKC-δ (1 : 500, rabbit antibodies, Beijing Biosynthesis Biotechnology Co. Ltd, China), p53 (1 : 500, rabbit antibodies, Beijing Biosynthesis Biotechnology Co. Ltd, China), p21cip1 (1 : 500, rabbit antibodies, Beijing Biosynthesis Biotechnology Co. Ltd, China), p27kip1 (1 : 500, rabbit antibodies, Beijing Biosynthesis Biotechnology Co. Ltd, China), E2F-1 (1 : 500, rabbit antibodies, Beijing Biosynthesis Biotechnology Co. Ltd, China) and β-actin [1 : 1000, rabbit antibodies, Cell Signaling Technology (CST), USA] overnight at 4 °C. After washing three times with TBST for 10 min each time, the PVDF membranes were incubated with an anti-rabbit IgG secondary antibody [1 : 5000, Immunology Consultants Laboratory (ICL), USA] for 1 h. Then the PVDF membranes were washed three times for 10 min each time using TBST. The enhanced chemiluminescence reagent (Pierce, USA) was used to detect the antibody-bound proteins.

Statistical analysis

SPSS (version 18.0, IBM) was used to analyze the data. The significant difference between all the groups was used and a one-way analysis of variance (ANOVA) was used to test this, and the significant differences between groups used least significant difference multiple range tests to assess this. All the values were expressed as the mean ± standard error (SE), and p < 0.05 were considered to be significant.

Results

Effect of endosulfan on cell apoptosis in vivo

In the in vivo experiment, a brown nucleus indicated apoptosis of cells and a blue nucleus indicated normal cells. The present results revealed that the number of brown nuclei in groups treated with endosulfan were significantly more than those in the control group (Fig. 1A). This result indicated that endosulfan obviously induced apoptosis of cells.

Fig. 1. Effect of endosulfan on apoptosis and the MDA content of spermatogenic cells in testicular tissue. A: Effect of endosulfan on the apoptosis of a spermatogenic cell in testicular tissue. Control group (a), 1 mg kg–1 endosulfan group (b), 5 mg kg–1 endosulfan group (c), 10 mg kg–1 endosulfan group (d). After exposure to various concentrations of endosulfan for 21 days, the changes in spermatogenic cells of testicular tissue were visualized at 400× magnification using a compound light microscope. In the images, the white arrow points to the brown nuclei which indicate the apoptotic cells. B: Effect of endosulfan on the MDA content of spermatogenic cell in testicular tissue. All the values are expressed as the mean ± SE. *p < 0.05 versus the control group.

Fig. 1

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Effects of endosulfan on MDA levels in testes

In the in vivo experiment, oxidative stress was detected by assessing the MDA levels in the testes. With increased endosulfan concentration, the MDA level gradually increased. The MDA level in the 1, 5 and 10 mg kg–1 endosulfan treated groups were more than that in the control group (Fig. 1B).

Effects of endosulfan on expression of cell cycle relative proteins in testes

This research revealed that the expression of PKC-δ, p53, p21cip1 and p27kip1 in testicular tissue increased gradually with the elevation of the endosulfan concentration (Fig. 2A and B). Compared to the control group, the expression of PKC-δ was obviously promoted in the 1, 5 and 10 mg kg–1 endosulfan groups, the expression of p27kip1 increased remarkably in the 5 and 10 mg kg–1 endosulfan group, and the expression of p53 and p21cip1 was apparently activated in the 10 mg kg–1 endosulfan group, whereas the expression of E2F-1 was obviously decreased in the 10 mg kg–1 endosulfan group (Fig. 2B).

Fig. 2. Effects of endosulfan on the expression of cell cycle relative proteins of spermatogenic cells in testicular tissue. A: Effect of endosulfan on protein expression of PKC-δ, p53, p21cip1, p27kip1 and E2F-1. β-Actin was used as an internal control to monitor the effect of equal loading. B: Semi-quantitative analysis of the effects of endosulfan on protein expressions of PKC-δ, p53, p21cip1, p27kip1 and E2F-1 expressed as the relative amount of grey level to β-actin in the same lane. All values were expressed as the mean ± SE. *p < 0.05 versus the control group.

Fig. 2

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Effects of endosulfan on the viability and integrity of the cell membrane in GC-1 spg cells

Treatment with endosulfan lead to decreased cell viability with increased concentration and extended time in different groups (p < 0.05) (Table 1). Meanwhile, the LDH activity increased in the groups treated with 6, 12 and 24 μg ml–1 endosulfan (Fig. 3C). These results indicated that treatment with endosulfan lead to decreased cell viability and damage of the cell membrane.

Table 1. Effect of endosulfan on percentage viability of GC-1 spg cells.

Endosulfan (μg ml–1) Percentage viability (%)
6 hours 12 hours 24 hours 48 hours
0 100 100 100 100
6 96.6 ± 0.4a 91.5 ± 0.4a 87.5 ± 0.3a 66.2 ± 0.4a
12 92.6 ± 0.6a,b 85.3 ± 0.3a,b 81.2 ± 0.8a,b 49.2 ± 0.3a,b
24 87.5 ± 0.3a,b,c 79.4 ± 0.6a,b,c 67.7 ± 1.3a,b,c 15.8 ± 0.1a,b,c
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Fig. 3. Effects of endousulfan on apoptosis and the activity of LDH of GC-1 spg cells. A: The apoptosis of GC-1 spg cells measured by staining with Annexin V-FITC after treatment with 0 (A1), 6 (A2), 12 (A3) and 24 (A4) μg ml–1 of endosulfan for 24 h. The lower left quadrant of the histograms shows the viable cells, the lower right quadrant represents the early apoptosis cells, the upper right represents the late-stage apoptotic cells, and the upper left quadrant represents the non-viable cells. The apoptosis rate = (Q2 + Q4)/(Q1 + Q2 + Q3 + Q4) × 100. B: Apoptosis rate of GC-1 spg cells in groups with different concentrations of endosulfan treatment. All values are expressed as the mean ± SE. The values with complete different superscript letters are significantly different among the various dose groups (p < 0.05). C: The activity of LDH of GC-1 spg cells in groups with different concentrations of endosulfan treatment. All values are expressed as the mean ± SE. The values with different superscript letters were significantly different among the various dose groups (P < 0.05).

Fig. 3

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Effects of endosulfan on the apoptosis in GC-1 spg cells

Endosulfan had a significant effect on the apoptosis in GC-1 spg cells in the in vitro experiment (p < 0.05). The apoptosis of GC-1 spg cells in 12 and 24 μg ml–1 endosulfan treated groups was greater than that in the control group (Fig. 3A and B).

Effects of endosulfan on ROS levels in GC-1 spg cells

In the in vitro experiment, oxidative stress was evaluated by measuring the ROS levels in GC-1 spg cells. The ROS levels of GC-1 spg cells in the 6, 12 and 24 μg ml–1 endosulfan treated groups were higher than that in control group (p < 0.05) (Fig. 4B).

Fig. 4. Effects of endosulfan on 8-OHdG and ROS in GC-1 spg cells. A: Variable nuclear and cytoplasmic expression of 8-OHdG in groups exposed to 0 (a), 6 (b), 12 (c) and 24 (d) μg ml–1 of endosulfan. (The arrows indicate the area of 8-OHdG expression) (cells stained with immunohistochemistry stain (IHC) stain, 400×.) B: Intracellular ROS levels in groups treated with 0, 6, 12 and 24 μg ml–1 of endosulfan were determined using flow cytometry with 2′,7′-dichlorofluorescin diacetate (DCFH-DA). The mean fluorescent intensity indicates ROS generation in GC-1 spg cells exposed to different concentrations of endosulfan. All the values are expressed as the mean ± SE. The values with completely different superscript letters were significantly different among various dose groups (p < 0.05).

Fig. 4

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Effect of endosulfan on DNA damage in GC-1 spg cells

Immunocytochemistry studies revealed that the expression of 8-oxo-2′-deoxyguanosine (8-OhdG) in the nuclei and cytoplasm of cells increased greatly in the groups exposed to 12 and 24 μg ml–1 endosulfan, whereas there were no significant changes in the group exposed to 6 μg ml–1 endosulfan. The results indicated that endosulfan could seriously damage DNA in GC-1 spg cells at concentrations from 12 to 24 μg ml–1 (Fig. 4B).

Effects of endosulfan on the protein expression of FasL and Fas in GC-1 spg cells

Immunocytochemistry showed that the number of cells showing positive FasL and Fas expression had obviously increased in the endosulfan-treated groups (Fig. 5A and B). Meanwhile, the western blotting and semi-quantitative analysis results revealed that the expression of FasL/Fas in endosulfan-treated groups had significant increases compared to the control group (p < 0.05) (Fig. 5C–E).

Fig. 5. Effects of endosulfan on the expressions of FasL/Fas in GC-1 spg cells. A–B: Expressions of FasL(A) and Fas(B) in groups treated with 0 μg ml–1 (a), 6 μg ml–1 (b), 12 μg ml–1 (c) and 24 μg ml–1 (d) endosulfan obtained using immunocytochemistry. (The arrows indicate the area of FasL expression) (IHC stain, 400×.) C: Effects of endosulfan on protein-expression of FasL and Fas in GC-1 spg cells. Western blot analysis of Fas and FasL expression in control and endosulfan-treated groups. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. D–E: Semi-quantitative analysis of the effect of endosulfan on the expression of FasL (D) and Fas (E) expressed as the relative amount of grey level to GAPDH in the same lane (mean ± SE). All the values are expressed as the mean ± SE. The values with different superscript letters were significantly different among various dose groups (p < 0.05).

Fig. 5

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Effects of endosulfan on the activities of Caspase-8 and Caspase-3 in GC-1 spg cells

The Caspase-8 activities of GC-1 spg cells in groups treated with 6, 12 and 24 μg ml–1 endosulfan were more than that in the control group (p < 0.05) (Fig. 6A). The Caspase-3 activity in the groups treated with 12 and 24 μg ml–1 endosulfan were significantly higher than that in the control group (p < 0.05) (Fig. 6B).

Fig. 6. The effects of endosulfan on Caspase-8 and Caspase-3 activity. A: Caspase-8. B: Caspase-3. All values are expressed as the mean ± SE. The values with different superscript letters were significantly different among various dose groups (p < 0.05).

Fig. 6

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Discussion

The physical condition of the spermatogenic cells has an influence on the quantity and quality of sperm. The present study was conducted to investigate the molecular mechanism of reproductive toxicity resulting from exposure to endosulfan. To begin with, the cell viability, cell membrane integrity and apoptosis rates of the GC-1 spg cells were measured. The results showed that endosulfan decreased the cell viability, damaged the cell membranes and increased the apoptosises of spermatogenic cells. To prove whether the oxidative stress played an important role in inducing the toxicity of spermatogenic cells to endosulfan, the levels of MDA and ROS were determined, and these biomarkers reflect the degree of oxidative stress.23,24 The present results showed that endosulfan could raise the MDA content in GC-1 spg cells and the ROS level in the testes. In other words, endosulfan induced oxidative stress. Takhshid et al.19 showed that endosulfan increased the MDA level of testis, too. A previous study by Yu et al. also found that the ROS levels in the groups treated with 6, 12 and 24 μg ml–1 of endosulfan were higher than that in the control group.25 Another study found that oxidative stress not only induced DNA base oxidation and direct deamination of the reactive oxygen, but also caused indirect base alkylation via lipid peroxidation.26 The present results showed that the higher concentration of endosulfan leads to severe damage on the DNA of spermatogenic cells. Research by Tao et al. obtained a similar result, which showed that the degree of DNA damage in tissues was related to the concentration of endosulfan.27

Cells were found to have a reversible cell cycle arrest resulting from transient or low levels of DNA damage, and a sustained cell cycle arrest followed high levels of DNA damage.28 The previous study by Guo et al. showed that the blockage of GC-1 spg cells by endosulfan had two phases, S and G2/M.29 Expression of p53 and p21cip1 could prevent cells entering into the M phase, whereas PKC-δ could regulate the level of p53 by increasing the transcription of p53.30 Expression of p21waf1/cip1and P27kipl were two important effector proteins in the negative regulation of the cell cycle mediated by PKC-δ.31 Expression of p21waf1/cip1 could cause cell cycle arrest in the G phase, and P27kipl may cause cell arrest in the G0/G1 phase and during apoptosis.32 A study indicated that p27kip1 could inhibit the activity of cyclin-dependent kinases (CDKs).33 CDKs could regulate E2F-1 by adjusting the Rb family.34 E2F-1 also has an effect on the regulation of the cell cycle.35 Therefore, E2F-1 is negatively correlated with the negative regulation pathway of the cell cycle mediated by PKC-δ. The present results showed that endosulfan led to the increase of PKC-δ, p53, p21cip1 and p27kip1 expression and a decrease of E2F-1 expression in spermatogenic cells. The results presented previously suggest that endosulfan activated the negative regulation pathway of the cell cycle mediated by PKC-δ, and that it could block the cell cycle by improving the expressions of PKC-δ, p53, p21cip1 and p27kip1, and by inhibiting the expression of E2F-1, which leads to further apoptosis in the spermatogenic cell.

Expression of p53 could be activated by DNA damage and mediates cell cycle arrest, leading to E2F-1-induced apoptosis.36,37 Furthermore, oxidative stress could induce apoptosis via a death receptor pathway.38 Fas/FasL could mediate the apoptosis of various cell types by the death receptor pathway after FasL binding with Fas receptors.39 The present results showed that endosulfan obviously increased the level of Fas and FasL, improved the activities of Caspase-8 and Caspase-3, and led to the apoptosis of spermatogenic cells. These results suggest that endosulfan could induce the apoptosis of spermatogonial cells by activating the death receptor pathway resulting from oxidative damage.

Conclusion

Endosulfan can decrease cell viability, damage cell membranes and lead to the apoptosis of spermatogonial cells, and also activate the protein expressions of PKC-δ, p53, p21cip1, p27kip1, improve the expressions of Fas, FasL, increase the activities of Caspase-8 and Caspase-3, inhibit the expressions of E2F-1, and induce oxidative stress and DNA damage. Endosulfan can lead to E2F-1-induced apoptosis of spermatogenic cells by activating the negative regulation factors of the cell cycle, and endosulfan might cause the apoptosis by death receptor pathway, which is activated by oxidative stress (Fig. 7).

Fig. 7. The possible mechanism of endosulfan effect on apoptosis of spermatogenic cells.

Fig. 7

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Conflict of interest

There are no conflicts of interest to declare.

Acknowledgments

This research was supported by the National Natural Science Foundation of China (no. 31172086).

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