Journal of Hazardous Materials 272 (2014) 75–82
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Sodium fluoride activates ERK and JNK via induction of oxidative stress to promote apoptosis and impairs ovarian function in rats Yanqing Geng 1 , Yiwen Qiu 1 , Xueqing Liu, Xuemei Chen, Yubin Ding, Shangjing Liu, Yi Zhao, Rufei Gao, Yingxiong Wang, Junlin He ∗ Laboratory of Reproductive Biology, School of Public Health and Management, Chongqing Medical University, Box 197, No. 1 Yixueyuan Road, Yuzhong District, 400016 Chongqing, PR China
h i g h l i g h t s • The toxicity of sodium fluoride (NaF) to female fertility was firstly investigated. • NaF exposure reduces E2 and P4 levels by inducing apoptosis in ovarian cells. • Oxidative stress plays a key role in NaF-induced ovarian dysfunction by activating the apoptotic ERK and JNK signaling pathways.
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Article history: Received 25 December 2013 Received in revised form 2 March 2014 Accepted 3 March 2014 Available online 18 March 2014 Keywords: Sodium fluoride Female reproduction Ovarian apoptosis Oxidative stress
a b s t r a c t The toxicity of sodium fluoride (NaF) to female fertility is currently recognized; however, the mechanisms are unclear. Previously, we reported a reduction in successful pregnancy rates, ovarian atrophy and dysfunction following exposure to NaF. The purpose of this study was to elucidate the underlying molecular mechanisms. Female Sprague-Dawley rats (10 rats/group) received 100 or 200 mg/L NaF in their drinking water for 6 months or were assigned to an untreated control group. Apoptotic indices and oxidative stress indicators in blood and ovarian tissue were analyzed following sacrifice. The results confirmed the NaF-induced ovarian apoptosis, with concomitant activation of oxidative stress. Further investigations in ovarian granular cells showed that exposure to NaF activated extracellular regulated protein kinase (ERK) and c-Jun NH2 kinase (JNK), disrupting the ERK and JNK signaling pathways, while p38 and PI3K remained unchanged. These data demonstrated that oxidative stress may play a key role in NaF-induced ovarian dysfunction by activating the apoptotic ERK and JNK signaling pathways. © 2014 Published by Elsevier B.V.
1. Introduction Fluoride exists naturally in varying amounts in soil, water and food. It has been widely used as an additive in toothpaste, mouthwash and drinking water to prevent dental caries. However, the range of benefits is limited and an excessive fluoride intake over a prolonged period of time could result in a serious public health problem [1]. There is increasing evidence of a close link between increasing environmental fluoride levels and decreasing fertility
∗ Corresponding author. Tel.: +86 023 68485926; fax: +86 023 68485008. E-mail addresses: [email protected] (Y. Geng), [email protected] (Y. Qiu), [email protected] (X. Liu), [email protected] (X. Chen), [email protected] (Y. Ding), [email protected] (S. Liu), [email protected] (Y. Zhao), gao ru [email protected] (R. Gao), [email protected] (Y. Wang), hejunlin [email protected] (J. He). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jhazmat.2014.03.011 0304-3894/© 2014 Published by Elsevier B.V.
rates [2,3]. Studies have verified the toxic effects of sodium fluoride (NaF) on the male reproductive system [4,5]. These include damage to testicular structures and a decrease in the spermatozoa capable of oocyte fertilization, which were found to be related to disorders in the oxidative system [6,7]. Further reports have found altered levels of reproductive hormones, fertility, histological structures and developmental outcomes in individuals exposed to relatively high concentrations of fluoride [7–9]. In a previous study, we reported a significant decrease in successful pregnancy rates in rats exposed to increasing concentrations of NaF. This was accompanied by a decrease in the secretions of the steroid hormones estrogen (E2 ) and progesterone (P4 ), and an increase in ovarian atrophy and abnormal follicular development [3]. The aim of this study was to identify the underlying molecular mechanisms by which NaF exposure causes ovarian dysfunction. Studies have shown that oxidative stress can have an impact on the production of granular cell steroid hormones, in particular E2 , which is an important predictor of ovarian response [10]. Fluoride
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has been found to induce toxicological effects in other systems through oxidative stress [11,12]. Therefore we hypothesized that oxidative stress might be involved in ovarian atrophy and dysfunction as a result of NaF exposure. In addition, increased cell apoptosis can lead to organ atrophy [13]. Apoptotic indices and oxidative stress indicators including ROS, SOD, CAT and GSHPx activities and MDA content were analyzed. Our results showed that apoptosis was enhanced and oxidative stress was increased in the ovaries of rats exposed to NaF. MAPK and PI3K signaling pathways play a key role in oxidative stress-mediated apoptosis. Thus, we examined the activation of PI3K and the MAPK family members, ERK, JNK and p38 in further study. The results revealed increased phosphorylation of extracellular regulated protein kinases (ERK) and c-Jun NH2 kinase (JNK). These are involved in NaF-induced ovarian apoptosis and activation of the MAPK signaling pathway, while p38 and PI3K remained unchanged following exposure to NaF. Taken together, our results suggested that ERK and JNK activated by oxidative stress participated in NaF-induced apoptosis in ovarian cells. 2. Materials and methods 2.1. Animals and administration of fluoride Female Sprague-Dawley rats were purchased from the Animal Facility of Chongqing Medical University, China [Certificate No.: SCXK(YU) 20070001]. The rats were housed in the specific pathogen-free facility of Chongqing Medical University under a 12:12 h light–dark cycle at a constant temperature (22 ± 2 ◦ C) and humidity (50%), with access to standard chow diet and water ad libitum. Thirty rats (8–10 weeks old; weighing 160–250 g) were randomly divided into three groups (10 rats/group) as follows: 100 mg/L NaF-treated group; 200 mg/L NaF-treated group; and an untreated control group. NaF (99% pure; Tianjin Guangfu Fine Chemical Research Institute, Tianjin, China) was used as the source of fluoride. The two NaF-treated groups had ad libitum access to distilled water containing 100 or 200 mg/L NaF, respectively; the control group received distilled water. Treatment was continued for 6 months. The rats were then sacrificed, serum samples and ovarian tissues were obtained. The ovarian tissue samples were fixed in formalin and embedded in paraffin (FFPE). The fluoride concentrations in the blood and tissues were measured using an ion selective electrode, as previously described [14]. 2.2. Primary granular cell culture Sixty rats (aged 3 weeks, weighing 50–60 g) received subcutaneous injections of PMSG (40 IU) and were sacrificed 48 h later. The ovaries were immediately removed and placed in phosphate buffered saline (PBS) under aseptic conditions, as previously described [15]. After washed, the ovaries were placed in Dulbecco Modified Eagle Medium (DMEM)/Hams F-12 medium (Gibco, Carlsbad, CA, USA) and the follicles were punctured under a dissecting microscope to release granular cells. The tissue suspension was transferred to a centrifuge tube and digested by 0.25% trypsin-0.02% EDTA in a 37 ◦ C, 5% CO2 incubator for 1 h. The granular cells were recovered by passing the cell suspension through a 200-micron stainless steel mesh, followed by centrifugation at 1000 × g for 5 min. The cells were resuspended in complete medium consisting of DMEM/F-12, 15% fetal bovine serum (FBS; Gibco) supplemented with 100 mg/mL streptomycin and 100 U/mL penicillin. Cells were seeded in culture flasks or culture plates and incubated at 37 ◦ C, 5% CO2 . Granular cells were identified by detecting the expression of follicle-stimulating hormone receptor (FSHR) by immunocytochemistry (IHC).
2.3. MTT assay and measurement of NaF half maximal inhibitory concentration (IC50 ) Ovarian granular cells were seeded in 96-well plates (3 × 104 /well) and treated with varying concentrations of NaF (0.01–0.8 mM) for 48 h. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was added (30 L/well) and the plates were then incubated for 4 h at 37 ◦ C. The purple formazan crystals were dissolved in 100 L dimethylsulfoxide (DMSO) and the plates were read using an automated microplate spectrophotometer at 570 nm. The NaF-treatment concentrations were selected by calculating the half maximal inhibitory concentration (IC50 ). 2.4. Detection of ovarian apoptosis by flow cytometry and DAPI staining Ovarian granular cells were seeded in 6-well culture plates (1 × 106 /well) and incubated at 37 ◦ C, 5% CO2 . The cells grew rapidly for the first 48 h and slowed down after 6 days. Therefore, we decided to add NaF (50 L) or the specific inhibitors on day 3 and collect the cells 48 h later for further experimentation. The cells were pelleted by centrifugation (1000 × g, 2 min) and resuspended in 1 mL PBS. For flow cytometry, the cells were incubated in the dark for 10 min with FITC-annexin V and propidium iodide (PI) before being analyzed. For DAPI staining, the cells were washed twice in PBS and then incubated for 5 min with 4 ,6diamidino-2-phenylindole (DAPI) diluted with 0.1% Triton X-100. Nuclear staining was observed under a fluorescence microscope. Cells exhibiting chromatin condensation and nuclear fragmentation were considered apoptotic. 2.5. Immunohistochemistry of ovarian tissue samples Immunohistochemistry (IHC) was carried out following standard procedures. Briefly, the FFPE ovarian tissue samples were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Antigen retrieval was carried out by placing the sections in sodium citrate buffer for 10 min at room temperature, followed by 15 min at 100 ◦ C in a microwave oven. Endogenous peroxidase was inhibited by incubation with 3% hydrogen peroxide for 10 min at room temperature. The sections were blocked in 10% normal goat serum for 30 min at 37 ◦ C, incubated with primary antibody at 4 ◦ C overnight, and then incubated with a biotinylated goat anti-rat IgG secondary antibody for 30 min at 37 ◦ C followed by streptavidin-conjugated horseradish peroxidase for 30 min at 37 ◦ C. Antibody staining was developed using a diaminobenzidine substrate for 5 min at room temperature. The sections were subsequently stained with hematoxylin. The following primary antibodies were used: Bcl (Millipore, Bedford, MA, USA), Bax (Millipore) and FSHR (Abcam, Cambridge, MA, USA). 2.6. Western blot analysis Total protein was extracted from the ovarian tissue samples. Equal volumes of protein were loaded onto a 10% SDSpolyacrylamide gel and separated by electrophoresis. The resulting protein bands were transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h in TBST buffer containing 5% non-fat milk. Immunoblotting was performed by incubating the membranes in 5% milk-TBST at 4 ◦ C overnight with primary antibody. The membranes were washed three times in TBST and incubated with secondary antibody for 1 h. The membranes were washed three times in TBST and the antibodies were detected by enhanced chemiluminescence (ECL) using the appropriate ECL reagents (Millipore). Quantification was performed by densitometric analysis using Quantity One software
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(Bio-Rad) and the results normalized to -actin. The following primary antibodies were used: Bcl, Bax, ERK, JNK and p-JNK (Millipore); p-ERK, p38, p-p38, PI3K and p-PI3K (Santa Cruz Biotec, Santa Cruz, CA, USA); Caspase-3 (Cell Signaling Technology, Danvers, MA, USA); and -actin (Sigma–Aldrich, St. Louis, MO, USA). Western blot analysis was also performed on ovarian granular cells that had been pretreated with specific inhibitors of ROS, ERK and JNK (50 M DPI, U0126 and SP600125, respectively). DMSO was used as vehicle for these inhibitors. Cell lysates were prepared for Western blotting after incubating the cells in the presence or absence of 0.4 mM NaF. The experiments were performed in triplicate. 2.7. TUNEL assay to identify apoptotic cells Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were performed using a TUNEL kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, the ovarian tissue sections were pretreated with 20 g/mL Proteinase K for 15 min at 37 ◦ C, washed in PBS, then incubated with TUNEL reaction mixture (label solution and enzyme solution) for 1 h at 37 ◦ C. After rinsing the sections three times in PBS for 5 min, 50 L Converter-POD was added to each section and they were incubated in a humidified chamber at 37 ◦ C for 30 min. The sections were rinsed three times in PBS and incubated with 50–100 L 3,3 -diaminobenzidine (DAB) substrate for 10 min at 15–25 ◦ C. After rinsing the sections a further three times in PBS, they were analyzed under a light microscope. Apoptotic cells were stained brown. 2.8. Detection of ROS, SOD, CAT and GSHPx activities and MDA content The ovarian tissue samples were homogenized in PBS and centrifuged at 1000 × g for 15 min to obtain the supernatant. NaF-treated primary granular cells were recovered, as described above, washed in PBS and centrifuged at 400 × g for 30 s at 4 ◦ C. The resulting cell pellet was lysed to release the protein. The activities of reactive oxygen species (ROS), superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase (CAT), and the malondialdehyde (MDA) content were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Yan Hui Biological Technology Co. Ltd., Shanghai, China), according to the manufacturer’s instructions. 2.9. Statistical analyses SPSS v. 13.0 software (SPSS Inc., Chicago, IL, USA) was employed for statistical analyses. Comparisons between different groups of variables were determined by analysis of variance (ANOVA), according to the general linear model procedure in SPSS. Differences were considered significant at P < 0.05. 3. Results 3.1. Validation of the NaF-toxicity model in rats The fluoride concentrations in blood taken from the rats of NaF-treated groups were significantly higher than those from the control group. This confirmed that the NaF-toxicity model had been successfully established (P < 0.05, Supplementary Fig. 1). 3.2. NaF induced apoptosis of rat ovarian cells in vivo In order to determine the underlying molecular mechanisms of ovarian atrophy and dysfunction following exposure to NaF [3], we
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examined the levels of apoptotic indicators in the ovarian tissue samples (Fig. 1). Compared to the control group, the TUNEL assay showed the number of apoptotic cells in the NaF-treated groups was increased (Fig. 1A). IHC showed that Bcl-2 and Bax were widely located in both follicular and luteal ovarian granular cells. Whereas Bcl-2 expression was significantly decreased, Bax expression was increased, in NaF-treated groups compared to the control group (Fig. 1B). Western blotting confirmed that the level of Bcl-2 was significantly lower in the NaF-treated group compared to the control group (P < 0.05); however no significant difference was observed in the expression of Bax between the three groups (Fig. 1C). Furthermore, Western blotting revealed that caspase-3, a downstream effector during apoptosis, was significantly increased in response to NaF-treatment (P < 0.05; Fig. 1C). These findings suggested that exposure to NaF-induced apoptosis in rat ovaries. 3.3. NaF caused oxidative stress in rat ovarian cells in vivo To investigate whether these abnormal functions were related to oxidative stress, we used ELISA to examine ROS, SOD, CAT and GSHPx activities and MDA content in the NaF-treated and untreated control groups. ROS activity was significantly increased in both of the NaF-treated groups (P < 0.05; Fig. 2A); SOD, CAT and GSHPx activities were reduced in the 200 mg/L NaF-treated group (P < 0.05; Fig. 2A); and MDA content was significantly higher in the 200 mg/L NaF-treated group compared to the control group (P < 0.05) but was not significantly different in the 100 mg/L NaF-treated group (Fig. 2B). These data indicated that NaF-induced oxidative stress in rat ovaries. 3.4. NaF activated ERK and JNK signaling pathways in ovarian cells in vivo To characterize the molecular mechanisms underlying NaFinduced cell apoptosis in ovaries, the activation of several MAPK family members (ERK, JNK and p38) and PI3K that play a central role in oxidative stress-mediated apoptosis were evaluated by Western blotting. The results revealed that NaF induced phosphorylation of JNK and ERK, however p38 and PI3K remained unchanged (Fig. 3). These results suggested that NaF may induce apoptosis in ovarian cells via activation of ERK and JNK signaling pathways. 3.5. NaF-induced oxidative stress and apoptosis in primary ovarian granular cells The steroid hormones E2 and P4 are synthesized in granular cells, therefore primary ovarian granular cells were selected for in vitro experimentation to further establish the relationship between NaF, oxidative stress and apoptosis in ovaries. The purity of the granular cells was >95%, as determined by IHC staining of FSHR (Supplementary Fig. 2A). The concentrations of NaF were set at 0.2 mM and 0.4 mM, based on the IC50 values determined by MTT (Supplementary Fig. 2B). FCM showed that the level of apoptosis in the NaF-treatment groups was significantly higher than that in the control group (P < 0.05; Fig. 4A). DAPI staining confirmed that NaFinduced apoptotic behavior in ovarian granular cells, as indicated by nuclear and chromatin condensation (Fig. 4B). We also found that low concentrations of NaF (0.1 mM) promoted cell apoptosis (Supplementary Fig. 2B), suggesting that different concentrations of NaF had different effects on cell behavior in ovaries. To validate the hypothesis that oxidative stress was involved in NaF-induced apoptosis in ovarian cells, the activities of ROS, SOD, CAT and GSHPx, and MDA content were examined by ELISA. The results were similar to the results in vivo: ROS activity was significantly increased and SOD, CAT and GSHPx activities were decreased in NaF-treated groups compared to the control group (P < 0.05;
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Fig. 1. NaF induces apoptosis in ovarian cells in rats in vivo. (A) TUNEL staining shows an increase in the number of apoptotic ovarian cells in the NaF-treated groups. Positive nuclei are stained brown. gc, granular cell; f, follicle; magnification 400×; scale bar, 50 m. (B) Immunohistochemistry shows expressions of Bcl-2 and Bax in the NaF-treated groups. Brown staining indicates positivity. NC, negative control; gc, granular cell; f, follicle; magnification 400×; scale bar, 50 m. (C) Western blotting shows a significant decrease in Bcl-2 and increase in caspase-3 protein expressions in the NaF-treated groups compared to the untreated control group. *P < 0.05.
Fig. 2. ELISA shows that NaF induces oxidative stress in ovarian cells in vivo. (A) Increased activities of ROS, SOD, CAT and GSHPx; (B) and increased MDA content in the NaF-treated groups compared to the untreated control group. *P < 0.05.
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Fig. 3. NaF activates ERK and JNK in ovarian cells. Western blotting shows an increase in the phosphorylation of ERK (p-ERK) and JNK (p-JNK) following NaF-treatment. *P < 0.05, relative to untreated control cells.
Fig. 5A); and MDA content was higher in granular cells exposed to NaF (P < 0.05; Fig. 5B). These data demonstrated that NaF could induce oxidative stress in primary ovarian granular cells. 3.6. Rescue of NaF-induced apoptosis in ovarian granular cells by inhibition of ERK and JNK signaling pathways To verify that NaF could induce apoptosis of ovarian cells via activation of ERK and JNK, we employed specific inhibitors of ERK and JNK (U0126 and SP600125, respectively). Western blotting showed that the expressions of phosphorylated ERK and JNK (p-ERK and
p-JNK, respectively) were significantly reduced by the addition of their corresponding inhibitors, and also by the addition of DPI, an inhibitor of ROS (P < 0.05; Fig. 6A). Furthermore, Bcl-2 expression was increased and Bax and casepase-3 were reduced in the NaFtreated groups following pretreatment with ERK and JNK inhibitors, either alone or in combination, and DPI (P < 0.05; Fig. 6B). Taken together, these results demonstrated that NaF-toxicity in ovarian cells could be rescued by suppressing the expression of ROS and activation of ERK and JNK, and indicated that ERK and JNK signaling pathways may be involved in the apoptosis of ovarian cells in response to NaF.
Fig. 4. Apoptosis is increased in primary ovarian granular cells following exposure to NaF. (A) Flow cytometric analysis shows an increase in the proportion of apoptotic cells following NaF-treatment. *P < 0.05, relative to untreated control cells. (B) The increase in the number of DAPI-stained nuclei also indicates an increase in apoptosis in NaF-treated cells. Apoptotic nuclei are indicated by yellow arrows; magnification 200×; scale bar, 100 m.
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Fig. 5. ELISA shows that NaF induces oxidative stress in primary ovarian granular cells. (A) Activation of ROS, SOD, CAT and GSHPx is increased in NaF-treated cells. (B) MDA content is increased following exposure to NaF. *P < 0.05, relative to untreated control cells; # P < 0.05, relative 0.2 mM NaF-treated cells.
4. Discussion Ovarian cells produce a number of hormones that are necessary for normal conceptus development and maintenance of pregnancy, including the ovarian steroids estrogen E2 and progesterone P4 . The levels of E2 and P4 define uterine receptivity, which
is essential for successful implantation of embryos in mammalian reproduction. [16]. E2 enhances the sensitivity of granular cells to follicle-stimulating hormone (FSH) and luteinizing hormone (LH), thereby increasing the biosynthesis of P4 in ovarian cells [17]. Furthermore, E2 modulates steroidogenesis, promotes granular cell proliferation and maintains follicular development [18,19]. As such,
Fig. 6. Specific inhibitors can rescue the toxicological effects of NaF in primary ovarian granular cells by partially blocking NaF-induced apoptosis. Ovarian granular cells were pretreated with DPI, U0126 or SP600125, which are specific inhibitors of ROS, ERK and JNK, respectively; (A) Western blotting shows a reduction in phosphorylated ERK (p-ERK) and JNK (p-JNK) indicating partial blocking of the ERK and JNK signaling pathways. (B) The increase in Bax and Caspase-3 expressions, and decrease in Bcl-2 expression indicates that pretreatment with these specific inhibitors can reduce the effects of NaF-toxicity. The data represent three independent experiments; *P < 0.05, relative to untreated control cells; # P < 0.05, relative to group treated without inhibitors.
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lower levels of E2 may be involved in the follicle developmental disorders that have been observed following NaF exposure. A decrease in the number of mature follicles will result in inhibition of ovulation and reduced fertility rates. In this study, we have provided evidence that NaF can induce apoptosis in ovarian cells. This would reduce E2 and P4 levels, leading to disorders of endometrial cell proliferation and differentiation, aberrant regulation of endometrial receptivity, and a decrease in embryo implantation rates. A variety of mechanisms have been proposed to explain fluoride-induced toxicity, including oxidative stress. Oxidative stress has been reported to be associated with reproductive impairment [20], and several reports have suggested that NaF can cause endometrial damage via oxidative stress [10,21]. In this study, we first showed the direct adverse effects of fluoride on ovaries via oxidative stress. Exposure to fluoride in rats inhibits the activity of SOD, GSHPx, and CAT, that would result in excessive production of reactive oxygen species (ROS). Increased O2 − concentration and its downstream consequences such as hydrogen peroxide, peroxynitrite, hydroxyl radicals could lead to oxidation of key mitochondrial proteins and ultimately mitochondrial dysfunction. These findings indicated that oxidative stress was involved in NaF-toxicity in ovaries and future investigations should be devoted to a deeper understanding of the molecular mechanisms underlying the effects of fluoride on mitochondrial gene expression and metabolism. In agreement with a previous report on the effects of NaF in ameloblasts, which showed that micromolar levels of fluoride could promote cell proliferation, while millimolar levels of fluoride inhibited cell proliferation [22], we found that different concentrations of NaF affected ovarian cell behavior differently. Whereas low concentrations of NaF (0.1 mM) promoted cell apoptosis. The underlying molecular mechanism by which oxidative stress, resulting from NaF exposure, can induce apoptosis in ovarian cells is unclear. Reports have suggested that oxidative stress might induce apoptosis via regulation of PI3K/Akt and MAPK signaling pathways [23–27]. Therefore we examined the activation of PI3K and the MAPK family members, ERK, JNK and p38, in ovarian granular cells. In agreement with these earlier reports, NaF activated MAPK signaling pathways by promoting phosphorylation JNK and ERK, but not p38 and
Fig. 7. Schematic model of NaF-induced toxicity in ovarian cells. NaF induces oxidative stress; which promotes production of ROS and generates MDA, and reduces expression of GSHPx, CAT and SOD; the ERK and JNK pathways are activated; Bcl2 expression is decreased and Bax expression is increased; triggering the caspase cascade to promote apoptosis.
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PI3K. These effects could be partially blocked by pretreatment with specific ERK inhibitor and JNK inhibitors. Furthermore, these intracellular events were also reversed by DPI, a ROS inhibitor. These results implied that ERK and JNK are upstream kinases implicated in NaF-induced ovarian apoptosis. Based on these results, a hypnotized pathway is schematically shown in Fig. 7. In conclusion, this study has shown that NaF can cause ovarian dysfunction by inducing cell apoptosis. Furthermore, oxidative stress played a key role in NaF-induced apoptosis via activation of ERK and JNK signaling. These findings could contribute to the prevention of reduced female fertility resulting from fluoride toxicity. Acknowledgment This work was supported by grants from the National Natural Science Foundation of China [30973195 and 31271246]. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2014. 03.011. References [1] S. Chouhan, S.J. Flora, Effects of fluoride on the tissue oxidative stress and apoptosis in rats: biochemical assays supported by IR spectroscopy data, Toxicology 254 (2008) 61–67. [2] S.C. Freni, Exposure to high fluoride concentrations in drinking water is associated with decreased birth rates, J. Toxicol. Environ. Health 42 (1994) 109–121. [3] Y. Zhou, et al., Effects of sodium fluoride on reproductive function in female rats, Food Chem. Toxicol. 56 (2013) 297–303. [4] Z. Sun, et al., Effects of sodium fluoride on hyperactivation and Ca2+ signaling pathway in sperm from mice: an in vivo study, Arch. Toxicol. 84 (2010) 353–361. [5] D. Ortiz-Perez, et al., Fluoride-induced disruption of reproductive hormones in men, Environ. Res. 93 (2003) 20–30. [6] Y.H. Xiao, et al., Effect of endemic fluoride poisoning caused by coal burning on the oxidative stress in rat testis, Zhongguo Yi Xue Ke Xue Yuan Xue Bao 33 (2011) 357–361. [7] J.A. Izquierdo-Vega, M. Sanchez-Gutierrez, L.M. Del Razo, Decreased in vitro fertility in male rats exposed to fluoride-induced oxidative stress damage and mitochondrial transmembrane potential loss, Toxicol. Appl. Pharmacol. 230 (2008) 352–357. [8] V. Dhar, M. Bhatnagar, Physiology and toxicity of fluoride, Indian J. Dent. Res. 20 (2009) 350–355. [9] T.F. Collins, et al., Developmental toxicity of sodium fluoride measured during multiple generations, Food Chem. Toxicol. 39 (2001) 867–876. [10] M. Appasamy, et al., Evaluation of the relationship between follicular fluid oxidative stress, ovarian hormones, and response to gonadotropin stimulation, Fertil. Steril. 89 (2008) 912–921. [11] N.I. Agalakova, G.P. Gusev, Fluoride induces oxidative stress and ATP depletion in the rat erythrocytes in vitro, Environ. Toxicol. Pharmacol. 34 (2012) 334–337. [12] V.K. Bharti, R.S. Srivastava, Fluoride-induced oxidative stress in rat’s brain and its amelioration by buffalo (Bubalus bubalis) pineal proteins and melatonin, Biol. Trace Elem. Res. 130 (2009) 131–140. [13] S. Takahashi, et al., Involvement of apoptosis and proliferation of acinar cells in atrophy of rat parotid glands induced by liquid diet, J. Mol. Histol. 43 (2012) 761–766. [14] L.M. Del Razo, et al., Fluoride levels in well-water from a chronic arsenicism area of Northern Mexico, Environ. Pollut. 80 (1993) 91–94. [15] T.N. Lovekamp, B.J. Davis, Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript levels and estradiol production in cultured rat granulosa cells, Toxicol. Appl. Pharmacol. 172 (2001) 217–224. [16] H. Wang, S.K. Dey, Roadmap to embryo implantation: clues from mouse models, Nat. Rev. Genet. 7 (2006) 185–199. [17] T.H. Welsh Jr., L.Z. Zhuang, A.J. Hsueh, Estrogen augmentation of gonadotropinstimulated progestin biosynthesis in cultured rat granulosa cells, Endocrinology 112 (1983) 1916–1924. [18] R.L. Robker, J.S. Richards, Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1, Mol. Endocrinol. 12 (1998) 924–940. [19] A.E. Drummond, J.K. Findlay, The role of estrogen in folliculogenesis, Mol. Cell. Endocrinol. 151 (1999) 57–64. [20] R.J. Aitken, et al., Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa, Biol. Reprod. 59 (1998) 1037–1046.
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Sodium fluoride activates ERK and JNK via induction of oxidative stress to promote apoptosis and impairs ovarian function in rats Yanqing Geng 1 , Yiwen Qiu 1 , Xueqing Liu, Xuemei Chen, Yubin Ding, Shangjing Liu, Yi Zhao, Rufei Gao, Yingxiong Wang, Junlin He ∗ Laboratory of Reproductive Biology, School of Public Health and Management, Chongqing Medical University, Box 197, No. 1 Yixueyuan Road, Yuzhong District, 400016 Chongqing, PR China
h i g h l i g h t s • The toxicity of sodium fluoride (NaF) to female fertility was firstly investigated. • NaF exposure reduces E2 and P4 levels by inducing apoptosis in ovarian cells. • Oxidative stress plays a key role in NaF-induced ovarian dysfunction by activating the apoptotic ERK and JNK signaling pathways.
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Article history: Received 25 December 2013 Received in revised form 2 March 2014 Accepted 3 March 2014 Available online 18 March 2014 Keywords: Sodium fluoride Female reproduction Ovarian apoptosis Oxidative stress
a b s t r a c t The toxicity of sodium fluoride (NaF) to female fertility is currently recognized; however, the mechanisms are unclear. Previously, we reported a reduction in successful pregnancy rates, ovarian atrophy and dysfunction following exposure to NaF. The purpose of this study was to elucidate the underlying molecular mechanisms. Female Sprague-Dawley rats (10 rats/group) received 100 or 200 mg/L NaF in their drinking water for 6 months or were assigned to an untreated control group. Apoptotic indices and oxidative stress indicators in blood and ovarian tissue were analyzed following sacrifice. The results confirmed the NaF-induced ovarian apoptosis, with concomitant activation of oxidative stress. Further investigations in ovarian granular cells showed that exposure to NaF activated extracellular regulated protein kinase (ERK) and c-Jun NH2 kinase (JNK), disrupting the ERK and JNK signaling pathways, while p38 and PI3K remained unchanged. These data demonstrated that oxidative stress may play a key role in NaF-induced ovarian dysfunction by activating the apoptotic ERK and JNK signaling pathways. © 2014 Published by Elsevier B.V.
1. Introduction Fluoride exists naturally in varying amounts in soil, water and food. It has been widely used as an additive in toothpaste, mouthwash and drinking water to prevent dental caries. However, the range of benefits is limited and an excessive fluoride intake over a prolonged period of time could result in a serious public health problem [1]. There is increasing evidence of a close link between increasing environmental fluoride levels and decreasing fertility
∗ Corresponding author. Tel.: +86 023 68485926; fax: +86 023 68485008. E-mail addresses: [email protected] (Y. Geng), [email protected] (Y. Qiu), [email protected] (X. Liu), [email protected] (X. Chen), [email protected] (Y. Ding), [email protected] (S. Liu), [email protected] (Y. Zhao), gao ru [email protected] (R. Gao), [email protected] (Y. Wang), hejunlin [email protected] (J. He). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jhazmat.2014.03.011 0304-3894/© 2014 Published by Elsevier B.V.
rates [2,3]. Studies have verified the toxic effects of sodium fluoride (NaF) on the male reproductive system [4,5]. These include damage to testicular structures and a decrease in the spermatozoa capable of oocyte fertilization, which were found to be related to disorders in the oxidative system [6,7]. Further reports have found altered levels of reproductive hormones, fertility, histological structures and developmental outcomes in individuals exposed to relatively high concentrations of fluoride [7–9]. In a previous study, we reported a significant decrease in successful pregnancy rates in rats exposed to increasing concentrations of NaF. This was accompanied by a decrease in the secretions of the steroid hormones estrogen (E2 ) and progesterone (P4 ), and an increase in ovarian atrophy and abnormal follicular development [3]. The aim of this study was to identify the underlying molecular mechanisms by which NaF exposure causes ovarian dysfunction. Studies have shown that oxidative stress can have an impact on the production of granular cell steroid hormones, in particular E2 , which is an important predictor of ovarian response [10]. Fluoride
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has been found to induce toxicological effects in other systems through oxidative stress [11,12]. Therefore we hypothesized that oxidative stress might be involved in ovarian atrophy and dysfunction as a result of NaF exposure. In addition, increased cell apoptosis can lead to organ atrophy [13]. Apoptotic indices and oxidative stress indicators including ROS, SOD, CAT and GSHPx activities and MDA content were analyzed. Our results showed that apoptosis was enhanced and oxidative stress was increased in the ovaries of rats exposed to NaF. MAPK and PI3K signaling pathways play a key role in oxidative stress-mediated apoptosis. Thus, we examined the activation of PI3K and the MAPK family members, ERK, JNK and p38 in further study. The results revealed increased phosphorylation of extracellular regulated protein kinases (ERK) and c-Jun NH2 kinase (JNK). These are involved in NaF-induced ovarian apoptosis and activation of the MAPK signaling pathway, while p38 and PI3K remained unchanged following exposure to NaF. Taken together, our results suggested that ERK and JNK activated by oxidative stress participated in NaF-induced apoptosis in ovarian cells. 2. Materials and methods 2.1. Animals and administration of fluoride Female Sprague-Dawley rats were purchased from the Animal Facility of Chongqing Medical University, China [Certificate No.: SCXK(YU) 20070001]. The rats were housed in the specific pathogen-free facility of Chongqing Medical University under a 12:12 h light–dark cycle at a constant temperature (22 ± 2 ◦ C) and humidity (50%), with access to standard chow diet and water ad libitum. Thirty rats (8–10 weeks old; weighing 160–250 g) were randomly divided into three groups (10 rats/group) as follows: 100 mg/L NaF-treated group; 200 mg/L NaF-treated group; and an untreated control group. NaF (99% pure; Tianjin Guangfu Fine Chemical Research Institute, Tianjin, China) was used as the source of fluoride. The two NaF-treated groups had ad libitum access to distilled water containing 100 or 200 mg/L NaF, respectively; the control group received distilled water. Treatment was continued for 6 months. The rats were then sacrificed, serum samples and ovarian tissues were obtained. The ovarian tissue samples were fixed in formalin and embedded in paraffin (FFPE). The fluoride concentrations in the blood and tissues were measured using an ion selective electrode, as previously described [14]. 2.2. Primary granular cell culture Sixty rats (aged 3 weeks, weighing 50–60 g) received subcutaneous injections of PMSG (40 IU) and were sacrificed 48 h later. The ovaries were immediately removed and placed in phosphate buffered saline (PBS) under aseptic conditions, as previously described [15]. After washed, the ovaries were placed in Dulbecco Modified Eagle Medium (DMEM)/Hams F-12 medium (Gibco, Carlsbad, CA, USA) and the follicles were punctured under a dissecting microscope to release granular cells. The tissue suspension was transferred to a centrifuge tube and digested by 0.25% trypsin-0.02% EDTA in a 37 ◦ C, 5% CO2 incubator for 1 h. The granular cells were recovered by passing the cell suspension through a 200-micron stainless steel mesh, followed by centrifugation at 1000 × g for 5 min. The cells were resuspended in complete medium consisting of DMEM/F-12, 15% fetal bovine serum (FBS; Gibco) supplemented with 100 mg/mL streptomycin and 100 U/mL penicillin. Cells were seeded in culture flasks or culture plates and incubated at 37 ◦ C, 5% CO2 . Granular cells were identified by detecting the expression of follicle-stimulating hormone receptor (FSHR) by immunocytochemistry (IHC).
2.3. MTT assay and measurement of NaF half maximal inhibitory concentration (IC50 ) Ovarian granular cells were seeded in 96-well plates (3 × 104 /well) and treated with varying concentrations of NaF (0.01–0.8 mM) for 48 h. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was added (30 L/well) and the plates were then incubated for 4 h at 37 ◦ C. The purple formazan crystals were dissolved in 100 L dimethylsulfoxide (DMSO) and the plates were read using an automated microplate spectrophotometer at 570 nm. The NaF-treatment concentrations were selected by calculating the half maximal inhibitory concentration (IC50 ). 2.4. Detection of ovarian apoptosis by flow cytometry and DAPI staining Ovarian granular cells were seeded in 6-well culture plates (1 × 106 /well) and incubated at 37 ◦ C, 5% CO2 . The cells grew rapidly for the first 48 h and slowed down after 6 days. Therefore, we decided to add NaF (50 L) or the specific inhibitors on day 3 and collect the cells 48 h later for further experimentation. The cells were pelleted by centrifugation (1000 × g, 2 min) and resuspended in 1 mL PBS. For flow cytometry, the cells were incubated in the dark for 10 min with FITC-annexin V and propidium iodide (PI) before being analyzed. For DAPI staining, the cells were washed twice in PBS and then incubated for 5 min with 4 ,6diamidino-2-phenylindole (DAPI) diluted with 0.1% Triton X-100. Nuclear staining was observed under a fluorescence microscope. Cells exhibiting chromatin condensation and nuclear fragmentation were considered apoptotic. 2.5. Immunohistochemistry of ovarian tissue samples Immunohistochemistry (IHC) was carried out following standard procedures. Briefly, the FFPE ovarian tissue samples were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Antigen retrieval was carried out by placing the sections in sodium citrate buffer for 10 min at room temperature, followed by 15 min at 100 ◦ C in a microwave oven. Endogenous peroxidase was inhibited by incubation with 3% hydrogen peroxide for 10 min at room temperature. The sections were blocked in 10% normal goat serum for 30 min at 37 ◦ C, incubated with primary antibody at 4 ◦ C overnight, and then incubated with a biotinylated goat anti-rat IgG secondary antibody for 30 min at 37 ◦ C followed by streptavidin-conjugated horseradish peroxidase for 30 min at 37 ◦ C. Antibody staining was developed using a diaminobenzidine substrate for 5 min at room temperature. The sections were subsequently stained with hematoxylin. The following primary antibodies were used: Bcl (Millipore, Bedford, MA, USA), Bax (Millipore) and FSHR (Abcam, Cambridge, MA, USA). 2.6. Western blot analysis Total protein was extracted from the ovarian tissue samples. Equal volumes of protein were loaded onto a 10% SDSpolyacrylamide gel and separated by electrophoresis. The resulting protein bands were transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h in TBST buffer containing 5% non-fat milk. Immunoblotting was performed by incubating the membranes in 5% milk-TBST at 4 ◦ C overnight with primary antibody. The membranes were washed three times in TBST and incubated with secondary antibody for 1 h. The membranes were washed three times in TBST and the antibodies were detected by enhanced chemiluminescence (ECL) using the appropriate ECL reagents (Millipore). Quantification was performed by densitometric analysis using Quantity One software
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(Bio-Rad) and the results normalized to -actin. The following primary antibodies were used: Bcl, Bax, ERK, JNK and p-JNK (Millipore); p-ERK, p38, p-p38, PI3K and p-PI3K (Santa Cruz Biotec, Santa Cruz, CA, USA); Caspase-3 (Cell Signaling Technology, Danvers, MA, USA); and -actin (Sigma–Aldrich, St. Louis, MO, USA). Western blot analysis was also performed on ovarian granular cells that had been pretreated with specific inhibitors of ROS, ERK and JNK (50 M DPI, U0126 and SP600125, respectively). DMSO was used as vehicle for these inhibitors. Cell lysates were prepared for Western blotting after incubating the cells in the presence or absence of 0.4 mM NaF. The experiments were performed in triplicate. 2.7. TUNEL assay to identify apoptotic cells Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were performed using a TUNEL kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, the ovarian tissue sections were pretreated with 20 g/mL Proteinase K for 15 min at 37 ◦ C, washed in PBS, then incubated with TUNEL reaction mixture (label solution and enzyme solution) for 1 h at 37 ◦ C. After rinsing the sections three times in PBS for 5 min, 50 L Converter-POD was added to each section and they were incubated in a humidified chamber at 37 ◦ C for 30 min. The sections were rinsed three times in PBS and incubated with 50–100 L 3,3 -diaminobenzidine (DAB) substrate for 10 min at 15–25 ◦ C. After rinsing the sections a further three times in PBS, they were analyzed under a light microscope. Apoptotic cells were stained brown. 2.8. Detection of ROS, SOD, CAT and GSHPx activities and MDA content The ovarian tissue samples were homogenized in PBS and centrifuged at 1000 × g for 15 min to obtain the supernatant. NaF-treated primary granular cells were recovered, as described above, washed in PBS and centrifuged at 400 × g for 30 s at 4 ◦ C. The resulting cell pellet was lysed to release the protein. The activities of reactive oxygen species (ROS), superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase (CAT), and the malondialdehyde (MDA) content were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Yan Hui Biological Technology Co. Ltd., Shanghai, China), according to the manufacturer’s instructions. 2.9. Statistical analyses SPSS v. 13.0 software (SPSS Inc., Chicago, IL, USA) was employed for statistical analyses. Comparisons between different groups of variables were determined by analysis of variance (ANOVA), according to the general linear model procedure in SPSS. Differences were considered significant at P < 0.05. 3. Results 3.1. Validation of the NaF-toxicity model in rats The fluoride concentrations in blood taken from the rats of NaF-treated groups were significantly higher than those from the control group. This confirmed that the NaF-toxicity model had been successfully established (P < 0.05, Supplementary Fig. 1). 3.2. NaF induced apoptosis of rat ovarian cells in vivo In order to determine the underlying molecular mechanisms of ovarian atrophy and dysfunction following exposure to NaF [3], we
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examined the levels of apoptotic indicators in the ovarian tissue samples (Fig. 1). Compared to the control group, the TUNEL assay showed the number of apoptotic cells in the NaF-treated groups was increased (Fig. 1A). IHC showed that Bcl-2 and Bax were widely located in both follicular and luteal ovarian granular cells. Whereas Bcl-2 expression was significantly decreased, Bax expression was increased, in NaF-treated groups compared to the control group (Fig. 1B). Western blotting confirmed that the level of Bcl-2 was significantly lower in the NaF-treated group compared to the control group (P < 0.05); however no significant difference was observed in the expression of Bax between the three groups (Fig. 1C). Furthermore, Western blotting revealed that caspase-3, a downstream effector during apoptosis, was significantly increased in response to NaF-treatment (P < 0.05; Fig. 1C). These findings suggested that exposure to NaF-induced apoptosis in rat ovaries. 3.3. NaF caused oxidative stress in rat ovarian cells in vivo To investigate whether these abnormal functions were related to oxidative stress, we used ELISA to examine ROS, SOD, CAT and GSHPx activities and MDA content in the NaF-treated and untreated control groups. ROS activity was significantly increased in both of the NaF-treated groups (P < 0.05; Fig. 2A); SOD, CAT and GSHPx activities were reduced in the 200 mg/L NaF-treated group (P < 0.05; Fig. 2A); and MDA content was significantly higher in the 200 mg/L NaF-treated group compared to the control group (P < 0.05) but was not significantly different in the 100 mg/L NaF-treated group (Fig. 2B). These data indicated that NaF-induced oxidative stress in rat ovaries. 3.4. NaF activated ERK and JNK signaling pathways in ovarian cells in vivo To characterize the molecular mechanisms underlying NaFinduced cell apoptosis in ovaries, the activation of several MAPK family members (ERK, JNK and p38) and PI3K that play a central role in oxidative stress-mediated apoptosis were evaluated by Western blotting. The results revealed that NaF induced phosphorylation of JNK and ERK, however p38 and PI3K remained unchanged (Fig. 3). These results suggested that NaF may induce apoptosis in ovarian cells via activation of ERK and JNK signaling pathways. 3.5. NaF-induced oxidative stress and apoptosis in primary ovarian granular cells The steroid hormones E2 and P4 are synthesized in granular cells, therefore primary ovarian granular cells were selected for in vitro experimentation to further establish the relationship between NaF, oxidative stress and apoptosis in ovaries. The purity of the granular cells was >95%, as determined by IHC staining of FSHR (Supplementary Fig. 2A). The concentrations of NaF were set at 0.2 mM and 0.4 mM, based on the IC50 values determined by MTT (Supplementary Fig. 2B). FCM showed that the level of apoptosis in the NaF-treatment groups was significantly higher than that in the control group (P < 0.05; Fig. 4A). DAPI staining confirmed that NaFinduced apoptotic behavior in ovarian granular cells, as indicated by nuclear and chromatin condensation (Fig. 4B). We also found that low concentrations of NaF (0.1 mM) promoted cell apoptosis (Supplementary Fig. 2B), suggesting that different concentrations of NaF had different effects on cell behavior in ovaries. To validate the hypothesis that oxidative stress was involved in NaF-induced apoptosis in ovarian cells, the activities of ROS, SOD, CAT and GSHPx, and MDA content were examined by ELISA. The results were similar to the results in vivo: ROS activity was significantly increased and SOD, CAT and GSHPx activities were decreased in NaF-treated groups compared to the control group (P < 0.05;
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Fig. 1. NaF induces apoptosis in ovarian cells in rats in vivo. (A) TUNEL staining shows an increase in the number of apoptotic ovarian cells in the NaF-treated groups. Positive nuclei are stained brown. gc, granular cell; f, follicle; magnification 400×; scale bar, 50 m. (B) Immunohistochemistry shows expressions of Bcl-2 and Bax in the NaF-treated groups. Brown staining indicates positivity. NC, negative control; gc, granular cell; f, follicle; magnification 400×; scale bar, 50 m. (C) Western blotting shows a significant decrease in Bcl-2 and increase in caspase-3 protein expressions in the NaF-treated groups compared to the untreated control group. *P < 0.05.
Fig. 2. ELISA shows that NaF induces oxidative stress in ovarian cells in vivo. (A) Increased activities of ROS, SOD, CAT and GSHPx; (B) and increased MDA content in the NaF-treated groups compared to the untreated control group. *P < 0.05.
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Fig. 3. NaF activates ERK and JNK in ovarian cells. Western blotting shows an increase in the phosphorylation of ERK (p-ERK) and JNK (p-JNK) following NaF-treatment. *P < 0.05, relative to untreated control cells.
Fig. 5A); and MDA content was higher in granular cells exposed to NaF (P < 0.05; Fig. 5B). These data demonstrated that NaF could induce oxidative stress in primary ovarian granular cells. 3.6. Rescue of NaF-induced apoptosis in ovarian granular cells by inhibition of ERK and JNK signaling pathways To verify that NaF could induce apoptosis of ovarian cells via activation of ERK and JNK, we employed specific inhibitors of ERK and JNK (U0126 and SP600125, respectively). Western blotting showed that the expressions of phosphorylated ERK and JNK (p-ERK and
p-JNK, respectively) were significantly reduced by the addition of their corresponding inhibitors, and also by the addition of DPI, an inhibitor of ROS (P < 0.05; Fig. 6A). Furthermore, Bcl-2 expression was increased and Bax and casepase-3 were reduced in the NaFtreated groups following pretreatment with ERK and JNK inhibitors, either alone or in combination, and DPI (P < 0.05; Fig. 6B). Taken together, these results demonstrated that NaF-toxicity in ovarian cells could be rescued by suppressing the expression of ROS and activation of ERK and JNK, and indicated that ERK and JNK signaling pathways may be involved in the apoptosis of ovarian cells in response to NaF.
Fig. 4. Apoptosis is increased in primary ovarian granular cells following exposure to NaF. (A) Flow cytometric analysis shows an increase in the proportion of apoptotic cells following NaF-treatment. *P < 0.05, relative to untreated control cells. (B) The increase in the number of DAPI-stained nuclei also indicates an increase in apoptosis in NaF-treated cells. Apoptotic nuclei are indicated by yellow arrows; magnification 200×; scale bar, 100 m.
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Fig. 5. ELISA shows that NaF induces oxidative stress in primary ovarian granular cells. (A) Activation of ROS, SOD, CAT and GSHPx is increased in NaF-treated cells. (B) MDA content is increased following exposure to NaF. *P < 0.05, relative to untreated control cells; # P < 0.05, relative 0.2 mM NaF-treated cells.
4. Discussion Ovarian cells produce a number of hormones that are necessary for normal conceptus development and maintenance of pregnancy, including the ovarian steroids estrogen E2 and progesterone P4 . The levels of E2 and P4 define uterine receptivity, which
is essential for successful implantation of embryos in mammalian reproduction. [16]. E2 enhances the sensitivity of granular cells to follicle-stimulating hormone (FSH) and luteinizing hormone (LH), thereby increasing the biosynthesis of P4 in ovarian cells [17]. Furthermore, E2 modulates steroidogenesis, promotes granular cell proliferation and maintains follicular development [18,19]. As such,
Fig. 6. Specific inhibitors can rescue the toxicological effects of NaF in primary ovarian granular cells by partially blocking NaF-induced apoptosis. Ovarian granular cells were pretreated with DPI, U0126 or SP600125, which are specific inhibitors of ROS, ERK and JNK, respectively; (A) Western blotting shows a reduction in phosphorylated ERK (p-ERK) and JNK (p-JNK) indicating partial blocking of the ERK and JNK signaling pathways. (B) The increase in Bax and Caspase-3 expressions, and decrease in Bcl-2 expression indicates that pretreatment with these specific inhibitors can reduce the effects of NaF-toxicity. The data represent three independent experiments; *P < 0.05, relative to untreated control cells; # P < 0.05, relative to group treated without inhibitors.
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lower levels of E2 may be involved in the follicle developmental disorders that have been observed following NaF exposure. A decrease in the number of mature follicles will result in inhibition of ovulation and reduced fertility rates. In this study, we have provided evidence that NaF can induce apoptosis in ovarian cells. This would reduce E2 and P4 levels, leading to disorders of endometrial cell proliferation and differentiation, aberrant regulation of endometrial receptivity, and a decrease in embryo implantation rates. A variety of mechanisms have been proposed to explain fluoride-induced toxicity, including oxidative stress. Oxidative stress has been reported to be associated with reproductive impairment [20], and several reports have suggested that NaF can cause endometrial damage via oxidative stress [10,21]. In this study, we first showed the direct adverse effects of fluoride on ovaries via oxidative stress. Exposure to fluoride in rats inhibits the activity of SOD, GSHPx, and CAT, that would result in excessive production of reactive oxygen species (ROS). Increased O2 − concentration and its downstream consequences such as hydrogen peroxide, peroxynitrite, hydroxyl radicals could lead to oxidation of key mitochondrial proteins and ultimately mitochondrial dysfunction. These findings indicated that oxidative stress was involved in NaF-toxicity in ovaries and future investigations should be devoted to a deeper understanding of the molecular mechanisms underlying the effects of fluoride on mitochondrial gene expression and metabolism. In agreement with a previous report on the effects of NaF in ameloblasts, which showed that micromolar levels of fluoride could promote cell proliferation, while millimolar levels of fluoride inhibited cell proliferation [22], we found that different concentrations of NaF affected ovarian cell behavior differently. Whereas low concentrations of NaF (0.1 mM) promoted cell apoptosis. The underlying molecular mechanism by which oxidative stress, resulting from NaF exposure, can induce apoptosis in ovarian cells is unclear. Reports have suggested that oxidative stress might induce apoptosis via regulation of PI3K/Akt and MAPK signaling pathways [23–27]. Therefore we examined the activation of PI3K and the MAPK family members, ERK, JNK and p38, in ovarian granular cells. In agreement with these earlier reports, NaF activated MAPK signaling pathways by promoting phosphorylation JNK and ERK, but not p38 and
Fig. 7. Schematic model of NaF-induced toxicity in ovarian cells. NaF induces oxidative stress; which promotes production of ROS and generates MDA, and reduces expression of GSHPx, CAT and SOD; the ERK and JNK pathways are activated; Bcl2 expression is decreased and Bax expression is increased; triggering the caspase cascade to promote apoptosis.
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PI3K. These effects could be partially blocked by pretreatment with specific ERK inhibitor and JNK inhibitors. Furthermore, these intracellular events were also reversed by DPI, a ROS inhibitor. These results implied that ERK and JNK are upstream kinases implicated in NaF-induced ovarian apoptosis. Based on these results, a hypnotized pathway is schematically shown in Fig. 7. In conclusion, this study has shown that NaF can cause ovarian dysfunction by inducing cell apoptosis. Furthermore, oxidative stress played a key role in NaF-induced apoptosis via activation of ERK and JNK signaling. These findings could contribute to the prevention of reduced female fertility resulting from fluoride toxicity. Acknowledgment This work was supported by grants from the National Natural Science Foundation of China [30973195 and 31271246]. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2014. 03.011. References [1] S. Chouhan, S.J. Flora, Effects of fluoride on the tissue oxidative stress and apoptosis in rats: biochemical assays supported by IR spectroscopy data, Toxicology 254 (2008) 61–67. [2] S.C. Freni, Exposure to high fluoride concentrations in drinking water is associated with decreased birth rates, J. Toxicol. Environ. Health 42 (1994) 109–121. [3] Y. Zhou, et al., Effects of sodium fluoride on reproductive function in female rats, Food Chem. Toxicol. 56 (2013) 297–303. [4] Z. Sun, et al., Effects of sodium fluoride on hyperactivation and Ca2+ signaling pathway in sperm from mice: an in vivo study, Arch. Toxicol. 84 (2010) 353–361. [5] D. Ortiz-Perez, et al., Fluoride-induced disruption of reproductive hormones in men, Environ. Res. 93 (2003) 20–30. [6] Y.H. Xiao, et al., Effect of endemic fluoride poisoning caused by coal burning on the oxidative stress in rat testis, Zhongguo Yi Xue Ke Xue Yuan Xue Bao 33 (2011) 357–361. [7] J.A. Izquierdo-Vega, M. Sanchez-Gutierrez, L.M. Del Razo, Decreased in vitro fertility in male rats exposed to fluoride-induced oxidative stress damage and mitochondrial transmembrane potential loss, Toxicol. Appl. Pharmacol. 230 (2008) 352–357. [8] V. Dhar, M. Bhatnagar, Physiology and toxicity of fluoride, Indian J. Dent. Res. 20 (2009) 350–355. [9] T.F. Collins, et al., Developmental toxicity of sodium fluoride measured during multiple generations, Food Chem. Toxicol. 39 (2001) 867–876. [10] M. Appasamy, et al., Evaluation of the relationship between follicular fluid oxidative stress, ovarian hormones, and response to gonadotropin stimulation, Fertil. Steril. 89 (2008) 912–921. [11] N.I. Agalakova, G.P. Gusev, Fluoride induces oxidative stress and ATP depletion in the rat erythrocytes in vitro, Environ. Toxicol. Pharmacol. 34 (2012) 334–337. [12] V.K. Bharti, R.S. Srivastava, Fluoride-induced oxidative stress in rat’s brain and its amelioration by buffalo (Bubalus bubalis) pineal proteins and melatonin, Biol. Trace Elem. Res. 130 (2009) 131–140. [13] S. Takahashi, et al., Involvement of apoptosis and proliferation of acinar cells in atrophy of rat parotid glands induced by liquid diet, J. Mol. Histol. 43 (2012) 761–766. [14] L.M. Del Razo, et al., Fluoride levels in well-water from a chronic arsenicism area of Northern Mexico, Environ. Pollut. 80 (1993) 91–94. [15] T.N. Lovekamp, B.J. Davis, Mono-(2-ethylhexyl) phthalate suppresses aromatase transcript levels and estradiol production in cultured rat granulosa cells, Toxicol. Appl. Pharmacol. 172 (2001) 217–224. [16] H. Wang, S.K. Dey, Roadmap to embryo implantation: clues from mouse models, Nat. Rev. Genet. 7 (2006) 185–199. [17] T.H. Welsh Jr., L.Z. Zhuang, A.J. Hsueh, Estrogen augmentation of gonadotropinstimulated progestin biosynthesis in cultured rat granulosa cells, Endocrinology 112 (1983) 1916–1924. [18] R.L. Robker, J.S. Richards, Hormone-induced proliferation and differentiation of granulosa cells: a coordinated balance of the cell cycle regulators cyclin D2 and p27Kip1, Mol. Endocrinol. 12 (1998) 924–940. [19] A.E. Drummond, J.K. Findlay, The role of estrogen in folliculogenesis, Mol. Cell. Endocrinol. 151 (1999) 57–64. [20] R.J. Aitken, et al., Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa, Biol. Reprod. 59 (1998) 1037–1046.
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