Nonylphenol-induced oxidative stress and cytotoxicity in testicular Sertoli cells

Reproductive Toxicology 22 (2006) 623–630

Nonylphenol-induced oxidative stress and cytotoxicity in testicular Sertoli cells Yi Gong, Xiao D. Han ∗ Immunology and Reproductive Biology Laboratory, Medical School, Nanjing University, Nanjing, Jiangsu 210093, PR China Received 19 May 2005; received in revised form 9 April 2006; accepted 14 April 2006 Available online 13 June 2006

Abstract Nonylphenol (NP) as an environment contaminant has been demonstrated to adversely affect male reproduction. The main objective of this study was to evaluate NP-induced oxidative stress and toxicity in testicular Sertoli cells. Sertoli cells were exposed to 10–40 ␮M NP for 24 h. Cell death and growth inhibition were observed by flow cytometric analysis and 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay, while fluorescein diacetate (FDA) and propidium iodide (PI) staining was used to examine the morphological changes following NP exposure. Subsequently, we found that short-term treatment (2 h) of NP caused intracellular accumulation of reactive oxygen species (ROS), which was evaluated by loading of 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) without visible morphological changes. Loss of mitochondrial membrane potential (MMP) was detected following 12 and 24 h treatment of NP and assessment by Rhodamine 123 (Rh123) staining. In addition, incubation with NP for 12 h also increased lipid peroxidation of Sertoli cells. These results indicated that low micromolar concentrations of NP induce an adverse oxidative stress in rat Sertoli cells. © 2006 Elsevier Inc. All rights reserved. Keywords: Nonylphenol; Sertoli cells; Reactive oxygen species; Mitochondrial membrane potential; Lipid peroxidation; Cell death

1. Introduction Nonionic surfactants possess specific physico-chemical characteristics, including relative ionic insensitivity and sorptive behavior, which lead to their extensive application in industry, processing technology and science, and usage in detergents. Alkylphenol ethoxylate (APEO), consisting of approximately 80% nonylphenol ethoxylate (NPEO), is a major group of nonionic surfactants [1,2]. The primary degradation product of NPEO, nonylphenol (NP) has been documented to appear in aquatic environment and sediment, where the concentration has approached mg/l and mg/kg level, respectively [3–5]. NP has weak estrogenic activity [6,7]. It has been demonstrated that NP could interfere with reproduction in fish, reptiles and mammals, and induce the cell death in gonads and changes to other reproductive parameters [8–10]. Previously, we found that male reproduction and testicular structure was disrupted in rats exposed to NP [11]; however, the precise mechanism of

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action is not clear. Some research has emphasized the estrogenic effect of NP, such as inducing the expression of estrogen receptor (ER) and inhibiting estrogen binding to ER, which might cause endocrine disruption [12,13]. Others studies, however, focused on cytotoxity. For example, NP could induce testicular cell death by inhibiting the activity of endoplasmic reticulum Ca2+ pumps [14]. Various environmental contaminants can induce oxidative stress by generating reactive oxygen species (ROS) such as hydrogen peroxide (H2 O2 ) and superoxide anion (O2 − ). ROS may initiate sequelae of reactions that damage cellular components culminating in cell death [15,16]. NP has been shown to produce oxidative stress, enhancing ROS generation in human blood neutrophils [17]. Furthermore, NP administration increased ROS level and lipid peroxidation and depressed the activity of antioxidant enzymes such as superoxide dismutase and glutathione reductase in rat testis [18]. We have taken the hypothesis that oxidative stress may partially be responsible for the reproductive toxicity following NP exposure. Sertoli cells are primary supporting cells for spermatogenesis and they contribute to the construction of the blood–testis barrier [19]. Thus, damage to Sertoli cells may

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lead to impairment of male reproduction. For these reasons, we selected primary rat Sertoli cells cultures to investigate the oxidative stress and cytotoxicity caused by NP.

37 ◦ C for 30 min. The absorbance was measured on an automated microplate reader (Bio-Rad, Japan) at 570 nm.

2.4. FDA and PI staining for morphologic evaluation 2. Material and methods 2.1. Chemicals and reagents NP (4-nonylphenol) with 98% analytical standard was from Tokyo Kasei Kogyo Co. (Tokyo, Japan). Dulbecco’s modified Eagle’s medium-Ham’s F-12 medium (DMEM-F12 medium), penicillin, streptomycin sulfate, trypsin, collagenase I, dimethyl sulphoxide (DMSO), fluorescein diacetate (FDA), propidium iodide (PI) and 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma–Aldrich Inc. (St. Louis, MO, USA). C8 H17 N2 O4 SNa (HEPES sodium salt) and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Amresco Inc. (Solon, OH, USA). Annexin V Apoptosis Kit, Cat. No. Sc-4252 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rhodamine 123 (Rh123) was bought from Calbiochem Inc. (La Jolla, CA, USA). The culture medium used was DMEM-F12 (1:1) supplemented with 4 mM glutamine, 15 mM Hepes, 6 mM l-(1)-lactic hemicalcium salt hydrate, 1 mM sodium pyruvate, antibiotics (final concentrations: penicillin, 100 IU/ml; streptomycin sulfate, 100 ␮g/ml) and 5% fetal bovine serum.

2.2. Isolation and culture of Sertoli cells

FDA, a none-fluorescent molecule can penetrate the cell membrane and is hydrolyzed by nonspecific esterases in viable cells producing green fluorescence that is retained in cytoplasm of intact cells. In the case of apoptotic and dead cells, fluorescence is decreased due to the lack of esterases and the leakage of FDA from the cells because of poor membrane integrity. PI, a nucleic acid binding dye, cannot penetrate the membrane of viable cells, but can enter cells readily during cell death process due to loss of membrane integrity. Therefore, FDA-PI staining is able to distinguish viable (FDA+ PI− ) and dead cells (FDA− PI+ ). In our study, Sertoli cells at the density of 2 × 105 ml−1 were seeded into 48-well plates and incubated for 24 h. Following treatment with various concentrations of NP for 24 h, cells were stained with 5 ␮g/ml PI and 4 ␮g/ml FDA and observed under the fluorescent microscopy (Nikon, Chiyoda-ku, Tokyo, Japan).

2.5. Flow cytometric assay Annexin V-PI staining combined with flow cytometry is commonly used to differentiate between cell apoptosis and necrosis. Briefly, 24 h after seeding Sertoli cells into a six-well plate, cells were exposed to various concentrations of NP for 24 h. Thereafter, cells were trypsinized and pelleted at 1500 rpm for 8 min followed by washing twice in PBS. For flow cytometry, cells were resuspended in assay buffer at the density of 106 cells/ml, and incubated at 5 ␮g/ml PI and 2 ␮g/ml Annexin V for 15 min in the dark. Finally, cell solution was diluted with fourfold volumes of assay buffer and analyzed by using a FACScan flow cytometer (Becton-Dickson, San Jose, CA, USA). All samples were treated gently to reduce the mechanical damage of cells.

Sprague–Dawley Rats were purchased from Nanjing Medical University and kept in accordance with NIH Guide for the Care and Use of Laboratory Animals. Sertoli cells were isolated from rats at the age of 20 days according to the method of Steinberger with some modifications [20]. Briefly, testes were decapsulated, minced and rinsed twice in phosphate-buffered saline (PBS), then digested with 0.25% trypsin at 37 ◦ C for 30 min. The isolated testicular fragments were centrifuged at 500 rpm and washed twice in PBS before further digestion in 0.1% collagenase for 30 min. Digested fragments were filtrated through a 100-mesh stainless steel filter. Cells were collected by centrifugation and then washed twice with PBS before being resuspended in DMEM-F12 medium. Finally, dispersed cells were seeded into culture flasks and maintained in a humidified atmosphere of 95% air, 5% CO2 at 34 ◦ C. Two days later, when Sertoli cells attached to the bottom of culture flasks, the supernatant was discarded. Then the purified Sertoli cells could quickly spread to form a monolayer in new medium.

Intracellular ROS were detected using DCFH-DA as described [16]. Inside cells, DCFH-DA was cleaved by esterases forming DCFH, which was in nonfluorescent form and was oxidized to fluorescent compound DCF by ROS. Following exposure to NP for 2 h, cells were loaded with 10 ␮M DCFH-DA for 10 min at 34 ◦ C and viewed under the fluorescence microscope. In addition, cellular fluorescence intensity was detected using a multi-detection microplate reader (Bio-TEK, Highland Park, Vermont, USA) at 488 nm.

2.3. Cell viability assay

2.7. Measurement of mitochondrial membrane potential

Cell viability was evaluated by the MTT test. Briefly, cells were plated on a 96-well culture plate at 2 × 104 cells/well in 100 ␮l culture medium. After incubation for 24 h, NP at various concentrations was added to each well. Then cells were cultivated for another 12 or 24 h, followed by the addition of 25 ␮l MTT solution (5 mg/ml) to each well and further incubation for 4 h. The supernatants were removed before adding 100 ␮l DMSO to dissolve the formazan crystal at

Mitochondrial membrane potential (MMP) was measured using Rh123, a positive charged molecule that can accumulate in energized mitochondria [21]. Decline of MMP will cause the leakage of Rh123 from mitochondria, resulting in the decline of fluorescence intensity. During the experiment, Sertoli cells treated with NP for 12 and 24 h were collected by trypsinization and centrifugation. Afterwards, cells in different groups were adjusted to the same density and

2.6. Measurement of intracellular ROS production

Fig. 1. MTT assay for cell viability after Sertoli cells were treated with 1, 10, 20, 30 and 40 ␮M NP for 12 h (A) and 24 h (B), respectively. Data are presented as mean ± S.D. (n = 6) and bars with different letters are significantly different (P < 0.05) from each other.

Y. Gong, X.D. Han / Reproductive Toxicology 22 (2006) 623–630 stained with 10 ␮g/ml Rh123 at 34 ◦ C in the dark for 30 min followed by rinsing in PBS. Fluorescence intensity was assessed using a multi-detection microplate reader at excitation wavelength of 488 nm and emission wavelength of 530 nm. In addition, Sertoli cells transplanted into 96-well plate at the same density were treated with NP for 12 h, stained with Rh123 and photographed under the fluorescent microscopy.

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Therefore, thiobarbituric acid-reactive substance (TBARS) assay is a method in common use to evaluate lipid peroxidation. Sertoli cells were treated with various concentrations of NP for 12 h, then trypsinized and centrifuged. The pellets triturated by ultrasonic were used to evaluate lipid peroxidation following the protocol of TBARS assay kit. The absorbance of the final supernatant was read at 532 nm on a multi-detection microplate reader.

2.8. Evaluation of lipid peroxidation 2.9. Statistical analysis Intracellular ROS can initiate a sequence of reactions resulting in the production of peroxidation compounds, such as malonic aldehyde (MDA), which can react with thiobarbituric acid (TBA) and form red products.

Results were shown as mean ± S.D. Statistical evaluation of the data was performed by using one-way analysis of variance (ANOVA) in SPSS followed

Fig. 2. Fluorescent pictures of Sertoli cells with FDA-PI staining. Cells exposed to NP for 24 h were stained with FDA (green) and PI (orange): (A) control, (B) 1 ␮M, (C) 10 ␮M, (D) 20 ␮M, (E) 30 ␮M, (F) 40 ␮M. (G) is higher-magnification pictures of Sertoli cells with FDA-PI staining. Arrow1 show FDA staining, while arrow 2 show PI staining within dead cells. Sertoli cells in the control show all viable cells, and an appreciable number of cells incubated in 20, 30 and 40 ␮M NP show orange within dead cells. Bar = 100 ␮m.

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by Student–Newman–Keuls post-test. P < 0.05 was accepted as the minimum level of significance.

3. Result 3.1. MTT assay for cell viability Cell viability was assessed by MTT assay following exposure to NP for 12 and 24 h (Fig. 1). NP significantly reduced cell viability at concentrations of 20 ␮M and higher; furthermore, the suppression increased with time. No significant difference was observed at low concentrations of NP (1 and 10 ␮M) relative to the control group. 3.2. Sertoli cell morphology FDA-PI staining was used to evaluate NP-induced morphologic changes on Sertoli cells. Fig. 2 shows representative fluorescent micrographs at each concentration of NP (Control, 1, 10, 20, 30 and 40 ␮M). NP treatment caused features of necrosis such as cell shrinkage and loss of membrane integrity as evidenced by the increase of PI staining (orange) as well as the decrease of FDA staining (green). This effect was strong at 20 ␮M and higher. Fig. 2G shows higher magnification, which more explicitly displays the FDA-PI staining.

3.3. Flow cytometric detection of dead cells Annexin V-PI staining combined with flow cytometry was used to study the effect of NP on Sertoli cell death. As shown in Fig. 3, higher concentration of NP yielded larger number of necrotic Sertoli cells, which were present as number (%) in upleft quadrant, and the effect was significant at the concentrations of 20 ␮M and higher. At lower concentrations of NP (1 and 10 ␮M), the percentage of dead cells also increased, but the effect was not as evident. 3.4. DCF assay for ROS The effect of NP on ROS generation, expressed as DCF fluorescence intensity, was determined in Sertoli cells. After exposure to NP for 2 h, although no distinct morphological changes were observed (Fig. 4), ROS increased at 40 ␮M NP. There were no significant differences between 0, 1, 10, 20, 30 ␮M groups, although a trend of increasing fluorescence intensity can be observed (Fig. 5). The experiment was repeated four times with similar results. 3.5. Effect of NP on mitochondrial membrane potential Loss of mitochondrial membrane potential (MMP), as measured by Rh123 staining, is shown in Fig. 6. The results

Fig. 3. NP-induced death of Sertoli cells as determined by flow cytometric assay. Cells transplanted into six-well plate were exposed to NP at concentrations of 1, 10, 20, 30 and 40 ␮M for 24 h. Numbers (%) in up-left quadrant represent the proportion of dead cells.

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Fig. 4. DCF fluorescent images of Sertoli cells upon exposure to NP. Cells were exposed to different concentrations of NP: (A) 0 ␮M, (B) 1 ␮M, (C) 10 ␮M, (D) 20 ␮M, (E) 30 ␮M and (F) 40 ␮M for 2 h, then loaded with DCFH-DA and viewed under fluorescence microscope. Bar = 50 ␮m.

revealed a suppressing effect of NP from 20 ␮M for 12 h, and 10 ␮M for 24 h. Fluorescence images of Sertoli cells treated with NP for 12 h also revealed the same trend (Fig. 7).

3.6. NP-induced lipid peroxidation of Sertoli cells Lipid peroxidation of Sertoli cells was investigated by TBARS assay. Both 30 and 40 ␮M NP markedly induced lipid peroxidation (Fig. 8); however, no significant increase of lipid peroxidation was detected at other concentrations of NP. 4. Discussion

Fig. 5. ROS levels expressed as DCF fluorescence intensity in Sertoli cells upon exposure to NP. Cells exposed to different concentrations of NP for 2 h were stained with DCFH-DA. Data are presented as mean ± S.D. (n = 6) and bars with different letters are significantly different (P < 0.05) from each other.

NP is a widespread aquatic contaminant that can accumulate in fish [22,23], which increases the latent risk of exposure to higher vertebrates via the food chain. Many reports have classified NP as hazardous to the health of human and animals, especially to male reproduction [11,24]. The present study demonstrated that NP was toxic to rat Sertoli cells. NP treatment resulted in the decrease of Sertoli cell viability and increased cell death. Furthermore, ROS generation, loss of mitochondrial membrane potential and increased lipid peroxidation were indicators of oxidative stress detected following NP exposure. It was well documented that exposure to NP exhibited inhibited cellular proliferation and led many kinds of cells to death

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Fig. 6. NP-induced decline of mitochondrial membrane potential. Mitochondrial membrane potential (indicated by fluorescence intensity of Rh123) of Sertoli cells after incubation in various concentrations of NP for 12 h (A) and 24 h (B) were measured. Data are presented as mean ± S.D. (n = 6) and bars with different letters are significantly different (P < 0.05) from each other.

[25,26]. Our study found that concentrations of NP (above 10 ␮M) were active toward Sertoli cell death; however, the mechanisms are still unknown. Some researchers hold the opinion that NP induces cell death by inhibiting endoplasmic reticulum Ca2+ pumps [14]. Others consider NP as the regulator of cellular cycle [25]. In our study, a slight increase of ROS production in Sertoli cells was observed after short-term (2 h)

treatment with NP but at the time of ROS determination no morphologic alteration or decrease of cell viability was observed. This suggested the increased ROS did not result from cell damage but rather from the proximate reaction of NP treatment. This finding is similar to other studies. For example, NP simulated the generation of ROS and impeded cell growth of E. coli mutant cells [27]. Another study reported NP-stimulated pro-

Fig. 7. Fluorescent images of NP-treated Sertoli cells with Rh123 staining. Sertoli cells at the same density were treated with 0 ␮M (A), 1 ␮M (B), 10 ␮M (C), 20 ␮M (D), 30 ␮M (E) and 40 ␮M (F) NP for 12 h and stained with Rh123 as mentioned in Section 2. Bar = 50 ␮m.

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Fig. 8. Effect of NP on lipid peroxidation of Sertoli cells. Values represent contents of MDA, the product of lipid peroxidation in each NP-treated group, which were expressed in the form of percentage of control. Data are presented as mean ± S.D. (n = 6) and bars with different letters are significantly different (P < 0.05) from each other.

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can also support the results of our research. The present study not only focused on the oxidative stress but also on the confirmation of the dose range that NP was functioning; furthermore, significance of the present study would be decreased if higher doses of NP were used, as NP level in the environment cannot approach so high. For all these reasons, only appropriate concentrations of NP were used in our study. In summary, direct exposure to NP can induce ROS generation in rat Sertoli cells and trigger a sequence of changes of the characteristics and functions of cellular components, such as loss of mitochondrial membrane potential and lipid peroxidation, subsequently depressing cellular viability and leading cells to death. It is likely that multiple mechanisms are at work, for only a slight increase of ROS was observed following NPtreatment. If all the effects were attributed to ROS, Sertoli cells should not have been so dramatically damaged by direct exposure. Acknowledgements

duction of ROS in rat epididymal sperm [28]. Therefore, both in prokaryotic cells and eukaryotic cells, NP can enhance ROS production. Recently, several studies demonstrated that ROS was a common inducer of cell death. Increased ROS may induce the peroxidation of cardiolipin in the mitochondrial inner membrane, which triggers dissociation of cytochrome c [29]. Once cytochrome c is released from mitochondria the cell is committed to die by activation of the apoptotic caspase cascade and nucleic DNA fragmentation, or necrosis due to collapse of the respiratory function, resulting from over-production of ROS and insufficient supply of ATP [30]. In this regard, the effect of higher concentrations of NP on Sertoli cell to dissipate mitochondrial polarization is consistent with other studies [31]. Moreover, it was reported that NP inhibited cellular respiration and proton extrusion, which were highly related to the function of mitochondria [32]. The mitochondrial respiratory chain is a major cellular site of ROS production [33]. ROS-induced attack to the components of mitochondria might in turn cause an increase of ROS level, resulting in the peroxidation of organellar membranes. From the outcomes of lipid peroxidation test following NP treatment, we demonstrated that high concentrations of NP (30–40 ␮M) increased lipid peroxidation. Lipid peroxidation may alter membrane characteristics and functions, leading to cell death. However, it is difficult to prove the causal relationship between lipid peroxidation and cell death, as they can affect each other. Many other chemicals shared the oxidative effect on cells. However, their concentrations used were very high. It was reported that 250 ␮M daidzein could induce great oxidative damage in human sperm and lymphocytes, while 5 mM halogenated methane caused hazardous oxidative stress in hepatocytes [33,34]. In comparison with these doses, the highest dose of NP chosen in our study was relatively small. The oxidative effect of low-dose fumonisin B1 induced a slight increase of oxidative stress; however, LDH release also increased [35]. This suggests a slight increase of oxidative stress can induce cytotoxicity, which

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