Apoptotic cell death in rat epididymis following epichlorohydrin treatment

Abstract

Epichlorohydrin (ECH) is an antifertility agent that acts both as an epididymal toxicant and an agent capable of directly affecting sperm motility. This study identified the time course of apoptotic cell death in rat epididymides after ECH treatment. Rats were administrated with a single oral dose of ECH (50 mg/kg). ECH-induced apoptotic changes were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and its related mechanism was confirmed by Western blot analysis and colorimetric assay. The TUNEL assay showed that the number of apoptotic cells increased at 8 h, reached a maximum level at 12 h, and then decreased progressively. The Western blot analysis demonstrated no significant changes in proapoptotic Bcl-2-associated X (Bax) and anti-apoptotic Bcl-2 expression during the time course of the study. However, phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) and phospho-c-Jun amino-terminal kinase (p-JNK) expression increased at 8–24 h. Caspase-3 and caspase-8 activities also increased at 8–48 h and 12–48 h, respectively, in the same manner as p-p38 MAPK and p-JNK expression. These results indicate that ECH induced apoptotic changes in rat epididymides and that the apoptotic cell death may be related more to the MAPK pathway than to the mitochondrial pathway.

Introduction

Epichlorohydrin (ECH) is a colorless liquid with an irritating chloroform-like odor that is used to manufacture epoxy resins, glycerin, coatings, adhesives, paints, varnishes, insecticides, and many other products. ¹ This chemical is one of the high-production-volume chemicals manufactured or imported at levels >1000 tons per year by at least one Organization for Economic Co-operation and Development member. Due to its increased production and widespread use, human exposure to ECH has steadily increased, which may result in adverse health impacts. ECH is rapidly and extensively absorbed following ingestion, inhalation, and dermal contact in the general population as well as in workers with specific occupations ² and damages the skin and the respiratory, gastrointestinal, and reproductive systems. ^{3 –5}

The epididymis plays an important role in sperm maturation, and its maturation is dependent on the luminal environment of the epididymis. ^6,7 Because the epididymis is intimately involved in spermatozoa acquiring motility and fertilizing ability, it is an ideal site for the action of antifertility agents and its dysfunction leads to defects in sperm maturation and fertility. ^8,9 The spermatotoxicity of ECH has been extensively studied in short- and long-term animal studies over the past several decades. ECH is an antifertility agent that acts both as an epididymal toxicant and an agent capable of directly affecting sperm motility. ^{10 –12} A single oral dose of alpha-chlorohydrin, a metabolite of ECH, at ≥25 mg/kg results in the deterioration of sperm function, possibly mediated by depleting ATP levels and inhibiting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity and white pustule, sperm granuloma, sloughed epithelial cell in the lumen and epithelial cell vacuolization, and disruption in the rat epididymis. ^13,14 We have demonstrated that the adverse effects of ECH on epididymal sperm and histology result from the induction of oxidative stress in rats. ¹⁵ Although ECH directly or indirectly affects the epididymis, whether ECH causes apoptotic changes in epididymis and whether apoptosis is involved in ECH-elicited spermatotoxicity has not been explored previously. The present study was conducted to establish whether apoptosis is implicated in ECH spermatotoxicity and to further study the potential mechanism involved in ECH-induced apoptotic cell death.

Materials and methods

Animals and environmental condition

A total of 36 sexually mature male Sprague–Dawley rats aged 9 weeks were purchased from Orient-Bio Inc. (Seoul, Korea) and used after 1 week of quarantine and acclimatization. Overall, 2 animals per cage were housed in a room maintained at a temperature of 23 ± 3°C and a relative humidity of 50 ± 10% with artificial lighting from 08:00 to 20:00. Commercial rodent chow (Samyang Feed Co., Wonju, Republic of Korea; 20% protein, 5% fat, 48.7% carbohydrate, and 4.5% fiber by weight) sterilized by radiation and sterilized tap water were provided ad libitum. The experimental design was approved by the Institutional Animal Care and Use Committee of Chonnam National University.

Test chemical and experimental groups

ECH (CAS No. 106-89-8; purity >99%) was purchased from Sigma-Aldrich Co. (St. Louis, Missouri, USA). ECH was dissolved in corn oil (Sigma-Aldrich Co.) and administered orally to rats. Overall, 30 male rats were randomly assigned to five groups of six rats each and were administered with a single oral dose of ECH (50 mg/kg) or its vehicle. In our previous study, we found that a 7-day repeated oral dose of 12.5 mg/kg/day ECH to rats increases the incidence of histopathological changes in the epididymis. ¹⁶ At a dose of 50 mg/kg/day, ECH decreases sperm motility and increases the incidence of sperm abnormalities and histopathological changes in the epididymis. Therefore, we chose 50 mg/kg as the ECH spermatotoxic dose in the present study. A total of 6 animals in each group were killed at 0 (control), 8, 12, 24, and 48 h after treatment.

In situ detection of fragmented DNA

At the scheduled necropsy, left caput epididymides of all rats were dissected and fixed in 10% neutral buffered formalin solution for 24 h. The fixed tissues were processed routinely and were embedded in paraffin, sectioned to 4 μm thickness, deparaffinized, and rehydrated using standard techniques. The level of DNA fragmentation was detected using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, which was performed according to the manufacturer’s instructions (ApopTag In Situ Apoptosis Detection Kit; Chemicon, Billerica, Massachusetts, USA). The number of TUNEL-positive cells was counted in 10 fields (×200) of similar size per slide in double-blinded manner and calculated as the total numbers of TUNEL-positive cells/10 fields. The results are presented as mean ± SD.

Western blotting analysis

For Western blot analysis, the epididymal tissues were lysed with a radioimmunoprecipitation assay buffer (RIPA lysis buffer) (Cell Signaling Technology, Lexington, Kentucky, USA) and centrifuged at 12,000×g at 4°C for 10 min to obtain the supernatant cellular proteins. Protein concentration was determined using the Bio-Rad Protein assay (Bio-Rad, Hercules, California, USA). Equal amounts of proteins from each sample were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Whatman, Maidenstone, UK), and blocked in blocking buffer (150 mM NaCl in 10 mM Tris, pH 7.5 containing 5% nonfat dry milk) for 1 h at room temperature. The membranes were incubated with primary rabbit antibodies against Bcl-2-associated X (Bax), Bcl-2, phospho-p38 mitogen-activated protein kinase (p-p38 MAPK), phospho-c-Jun amino-terminal kinase (p-JNK), or β-actin (Cell Signaling Technology, Beverly, Massachusetts, USA; 1:1000) for 18 h at 4°C, washed three times (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, and 0.1% Tween 20), incubated with HRP-conjugated secondary antibodies (1:2000) for 1 h at room temperature, washed three times, and then detected by enhanced chemiluminescence (Supersiganl West Pico, Pierce, Rockford, Illinois, USA). Proteins were quantified based on band density using TINA 20 Image software (Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany). Relative intensity was calculated by dividing the densities of respective proteins by β-actin density value. Values are presented as mean ± SD.

Determination of caspase-3 and caspase-8 activities

Caspase-3 and caspase-8 activities were measured in epididymal tissues with a colorimetric assay system (R&D Systems, Minneapolis, Minnesota, USA), according to the manufacturer’s instructions. Briefly, samples were lysed in 500 μl Cell Lysis Buffer (R&D Systems) on ice for 10 min and centrifuged at 10,000×g for 1 min. The supernatants were collected and protein concentrations were determined using the Bio-Rad Protein Assay. Protein lysates (100 μg) were incubated with 2× Reaction Buffer (R&D Systems). The specific peptide substrates used for each individual caspase were Asp-Glu-Val-Asp (DEAD) and Ile-Glue-Thr-Asp (IETD) labeled with p-nitroaniline (pNA) for caspase-3 and caspase-8, respectively. Reaction mixtures without epididymides extract were used as the negative control. Release of the pNA cleavage product was quantitated using a spectrophotometer (TECAN, Männedorf, Switzerland) at a wavelength of 405 nm.

Statistical analysis

Results are expressed as mean ± SD, and all statistical comparisons were made with a one-way analysis of variance followed by the Tukey–Kramer multiple comparison test. Differences with a p ≤ 0.05 were considered statistically significant.

Results

Effects of ECH on epididymal epithelial cell apoptosis

Administration of ECH to male rats produced apoptotic cell death in this time course study. As shown in Figure 1, male rats treated with a single oral dose of 50 mg/kg ECH showed a slight increase in the number of apoptotic cells in the proximal caput epididymis at 8 h, which reached a maximum level at 12 h (p < 0.01), decreased progressively at 24 h, and then recurrently increased at 48 h (p < 0.01) after treatment.

Figure 1.

Representative photographs of TUNEL assay performed on sections of epididymides from the control and treatment groups. Caput epididymides of (A) a control (0 hr), (B -E) 8, 12, 24, and 48 hr following exposure to ECH, respectively. (F) TUNEL-positive cell numbers in the ducts were quantified and expressed as means ± SD (n=6). Bar = 40 μm (4 μm section thickness, ×200).

Effects of ECH on mitochondrial-dependent and MAPKs-dependent pathways

Protein expression levels of Bax, Bcl-2, p-JNK, and p-p38 MAPK were analyzed by Western blotting to identify the apoptotic pathway involved in ECH-induced apoptotic cell death. Proapoptotic Bax and antiapoptotic Bcl-2 protein expression levels did not change during the time course Figure 2A. In contrast, p-p38 MAPK and p-JNK protein expression levels increased significantly at 8 h (p < 0.01), reached a peak level at 12 h (p < 0.01), and decreased progressively after 24 h following ECH treatment Figure 2B and C.

Figure 2.

Western blot analysis of Bax, Bcl-2, p-p38 MAPK and p-JNK in the epididymides of rats treated with ECH. Detection of β-actin expression was used as a loading control. The bar graphs show quantitative relative levels of (B) p-JNK and (C) p-p38 MAPK protein expression in ECH-treated rats. Values are means ± SD (n=6). ** P < 0.01 compared with the control group.

Effects of ECH on caspase-8 and caspase-3 activities

To further confirm these results, caspase-8 and caspase-3 activities were measured by colorimetric assay. As seen in Figure 3A and B, caspase-8 activity increased at 8 h, reached a maximum activity at 12 h (p < 0.01), decreased at 24 h (p < 0.05), and recurrently increased at 48 h (p < 0.01). Similarly, caspase-3 activity increased at 8 h (p < 0.01), showed a maximum activity at 12 h (p < 0.01), decreased progressively at 24 h (p < 0.05), and increased again at 48 h (p < 0.01).

Figure 3.

(A) Caspase-8 and (B) caspase-3 activities in epididymides treated with ECH. Values are means ± SD (n=6). * P < 0.05 compared with the control group. ** P < 0.01 compared with the control group.

Discussion

ECH acts as epididymal toxicant by inhibiting GAPDH activity in rat sperm and epididymis, followed by the depletion of sperm ATP. In our previous study, a 70-day repeated oral dose of ECH at ≥3.3 mg/kg/day caused adverse effects on epididymal sperm and histology by inducing oxidative stress and toxicity. ¹⁵ In other studies, ECH and alpha-chlorohydrin caused histopathological alterations in epididymis characterized by epithelial cell vacuolization and disruption. ^15,16 Although it is well established that ECH elicits toxic effects on epididymal structure and function, the mechanism of ECH-induced apoptotic cell death is still poorly understood. In the present study, we demonstrated that ECH treatment caused an increase in the number of apoptotic cells in the epididymides and that apoptotic cell death could be induced by concurrent activation of the p38 MAPK and JNK pathways but was not associated with the mitochondrial pathway.

Male rats treated with a single oral dose of ECH 50 mg/kg showed a slight increase in the number of apoptotic cells in the proximal caput epididymis at 8 h, which reached a maximum level at 12 h, and then decreased progressively at 24 h after treatment. As shown in Figure 1, ECH caused apoptosis of principal cell in epididymis. Principal cells secret proteins attributed to sperm maturation and possess androgen receptors associated with testosterone metabolism. ¹⁷ Therefore, it is considered that epididymal cell apoptosis observed in this study is involved in the deterioration of the epididymis by ECH treatment and affects the sperm maturation and motility.

We analyzed the apoptotic pathway and related protein expression in the epididymis by Western blot analysis to further understand the apoptotic cell death in ECH-induced spermatotoxicity. Apoptosis is a mechanistically driven form of cell death that is either developmentally regulated or activated in response to specific stimuli or various forms of cell injury. Apoptosis can be induced via two distinct signaling pathways such as the death receptor-mediated (extrinsic) or the mitochondrial-mediated (intrinsic) pathway. ^{18 –20} In the mitochondrial pathway, interactions between pro-apoptotic proteins and anti-apoptotic proteins play a role in mediating apoptotic signals. Bcl-2 blocks the release of cytochrome c from mitochondria in the apoptosis mitochondrial pathway, and Bax mediates the release of cytochrome c from mitochondria to the cytosol by forming permeabilization pores that trigger caspase-9 activation, followed by caspase-3 activation. ^19,20 The death receptor pathway is triggered by ligation signals on death receptors such as Fas receptor (FasR), tumor necrosis factor receptor (TNFR), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) leading to caspase-8 recruitment around the ligated receptors and proteolytic activation. Activated caspase-8 cleaves and activates caspase-3, which then initiates a series of events culminating in apoptosis. ¹⁸ MAPKs respond to extracellular stimuli, including tumor necrosis factor (TNF-α) and regulate various cellular activities, such as gene expression, mitosis, proliferation, and cell survival/apoptosis. ²¹ Three MAPK groups have been intensively studied, including extracellular signal-regulated kinase (ERK) that is responsive to mitogens such as growth factors, and JNK and p38 MAPK that are activated by pro-inflammatory cytokines and environmental stressors including TNF-α, interleukin-1, and chemotherapeutic drugs. ^{22 –25} In the present study, Bax, a member of the pro-apoptotic Bcl-2 family, and Bcl-2, a member of the anti-apoptotic family, protein expression levels did not change during the time course Figure 2A. In contrast, p-p38 MAPK and p-JNK expression levels increased significantly at 8 h, reached peak levels at 12 h, and decreased progressively after 24 h Figure 2B and C. The time course pattern of p-JNK and p-p38 MAPK protein expression was well correlated with that of apoptotic cell number, suggesting that activating the MAPK signaling pathway, particularly the p38 MAPK and JNK pathways, may be responsible for ECH-induced apoptosis in the epididymides and was not associated with the mitochondrial pathway.

We further analyzed caspase-8 and caspase-3 activities to confirm these results. Caspase-8 and caspase-3 activities increased at 8–48 h and 12–48 h after treatment, respectively Figure 3A and B, and showed a similar tendency as apoptotic cell number and p-p38 MAPK and p-JNK expression. Although it is unclear whether ECH activates the ERK pathway, these results suggest that the p38 MAPK and JNK pathways are involved in the activation of the upstream caspase cascade, resulting in caspase-3 activation as an apoptotic executioner. ECH has been classified as an epididymal toxicant and has either direct or indirect effects on the epididymal epithelium and epididymal spermatozoa causing sperm infertility. ^10,26 A single oral dose of ECH 70 mg/kg causes oligospermia, and epithelial disruption in the proximal caput epididymides concurrent with a decrease in sperm motility, without testicular effects. ²⁷ Considering above and our results, ECH-induced apoptosis is also partially related to deterioration of the epididymis and epididymal dysfunction, which is involved in the impaired acquisition of sperm motility and fertility.

Based on these findings, a single oral administration of ECH to male rats at 50 mg/kg elicited apoptotic changes in epididymal tissue, and these apoptotic changes may be related to activating the p38 MAPK and JNK pathways but not the mitochondrial pathway. However, further studies are needed to understand the precise mechanism of ECH toxicity.