Glycidamide inhibits progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells

Food and Chemical Toxicology xxx (2016) 1e8

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Glycidamide inhibits progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells Mingwei Li a, 1, Jianxia Sun b, 1, Feiyan Zou a, 1, Shun Bai a, Xinwei Jiang a, Rui Jiao a, Shiyi Ou a, Hui Zhang c, Zhijian Su a, Yadong Huang a, Weibin Bai a, * a

Department of Food Science and Engineering, Department of Developmental and Regenerative Biology, Biopharmaceutical R&D Center, Jinan University, Guangzhou, 510632, China Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou, 510006, China c China Rural Technology Development Center, Beijing, 100045, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 May 2016 Received in revised form 27 August 2016 Accepted 19 September 2016 Available online xxx

The food contaminant acrylamide (AA) is usually recognized as a probable human carcinogen. In addition, AA has also been found able to induce male infertility in animals. Interestingly, resent research work revealed that the toxic effect of AA on the ability of male reproduction in vivo may due to glycidamide (GA) which is the metabolite of AA. In this study, R2C Leydig cells was used to investigate the toxic effects of GA on progesterone production. GA caused dose-dependent inhibition on the cell growth, with IC25, IC50, and IC75 values found at 0.635, 0.872, and 1.198 mM, respectively. The results of single cell gel/Comet assay showed that GA significantly induced early-phase cell apoptosis, reduced progesterone production, as well as decreasing the protein expression of steroidogenic acute regulatory (StAR) in R2C cells. Furthermore, GA induced overproduction of intracellular reactive oxygen species (ROS), upregulated Bax expression, decreased mitochondrial membrane potential, and triggered mitochondria-mediated cell apoptosis. Consequently, the downstream effector caspase-3 was activated, resulting in Leydig cells apoptosis. Overall, our results showed that GA could damage R2C Leydig cells by the lesion of the ability of progesterone genesis and inducing cells apoptosis. © 2016 Published by Elsevier Ltd.

Keywords: Glycidamide R2C cells Progesterone ROS Apoptosis Mitochondrial membrane potential

1. Introduction Acrylamide (AA) is a chemical soluble in water and ethanol. AA is commonly used in the industry of synthesis of polyacrlamides, however, it was also discovered in various kinds of foods which have been treated with high temperature (Supplemental Fig. 1A) (Friedman, 2015). In humans, occupational exposure to AA has shown neurotoxic effect, and AA is classified as a probable human carcinogen (Pedreschi et al., 2014). Testicular toxicity has been reported in rodents exposed to AA with 5 mg/kg/day (Wang et al., 2010). . F344 rats were treated with 8 mg AA/kg body weight (bw)/day via the drinking water for 60 days. After sacrifice, the expression of several genes involved in oxidative stress and apoptosis were upregulated in testicular tissues (Mei et al., 2010). A fraction of ingested AA (50 mg/kg of body weight) is transformed by the monooxygenase CYP2E1 to GA (Supplemental Fig. 1B), which is

* Corresponding author. E-mail address: [email protected] (W. Bai). 1 These authors contributed equally for this work.

more reactive than AA due to its epoxide structure (Sumner et al., 1999). Thus, the epoxide GA is thought to be the ultimate genotoxic metabolite of AA. For instance, 1 mM GA administration in CYP2E1-deficient mice resulted in significantly reduced levels of male germ cell mutagenicity, micronuclei (MN) and GAeDNA adducts as compared to those observed in wild-type mice (Hansen et al., 2010). Given that covalent adduction of DNA by GA is a relevant genotoxic event, additional studies focusing on the mechanisms of GA-induced detrimental effects are fully warranted. Despite extensive researches focused on AA-induced reproductive toxicity, there is still a lack of information on the deleterious effects of GA on Leydig cells. A number of AA on animal toxicity studies (10 mg/kg of body weight/day) have shown that it exhibits reproductive toxicity in most in vivo assays (Camacho et al., 2012; Cengiz and Gunduz, 2013). GA is typically more cytotoxic than AA in vitro and in vivo, and increasing evidences suggest that GA plays a pivotal role in AA toxicity (Martins et al., 2007). Recent findings have shown that GA decreased testosterone secretion in cultured rat Leydig cells in vitro (Hansen et al., 2010). Previous investigators have demonstrated that steroids play fundamental roles in regulating mammalian

http://dx.doi.org/10.1016/j.fct.2016.09.035 0278-6915/© 2016 Published by Elsevier Ltd.

Please cite this article in press as: Li, M., et al., Glycidamide inhibits progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.09.035

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previously (Sun et al., 2014). Briefly, R2C cells were treated with GA and harvested and embedded in 0.8% low melting agarose on slides precoated with normal melting point agarose. Then, the slides were incubated in a lysis solution (2.5 mM NaCl, 0.1 M EDTA, 10 mM Tris, pH 10, 10% DMSO, and 1% Triton  100) for 1 h at approximately 5
C. Electrophoresis was performed at 25 V and 180 mA for 20 min in a freshly prepared alkaline buffer (0.3 M NaOH and 1 mM EDTA, pH 13.6). The slides were then placed in a horizontal electrophoresis tank (Bio-Rad, Hercules, CA, USA), exposed to 25 V for 15 min, washed twice with neutralization buffer (0.4 M Tris, pH 7.5), and dehydrated in ethanol for 5 min. After ethidium bromide staining, 50 randomly selected cells per coded slide were visualized under a fluorescent microscope and submitted to image analysis using CASP software. Both the Olive tail moment (OTM), defined as the product of the distance between the barycenters of the head and tail and the proportion of DNA in the tail, and the percentage of DNA in the comet tail were used to evaluate the extent of DNA damage in individual cells.

reproduction (Stout et al., 2010). Progesterone, synthesized from testosterone, is one of the main steroid hormones that manipulates reproductive function (Wiltbank et al., 2014). Leydig cells are the primary source of testosterone, and their differentiation in the testes is a signature event in the development of males (Wu et al., 2010; Sanderson, 2006). Steroid hormone synthesis is initiated by steroidogenic acute regulatory (StAR) protein, a key factor in the transfer of free cholesterol from the cytoplasm to the inner membrane of the mitochondria (Kim et al., 2011). The cAMP pathway is a major pathway involved in the regulation of steroidogenesis (Sun et al., 2013). GA has been shown to cause moderate testicular damage and consistently increase the activity of caspase 3 (Chen et al., 2013). This indicates that GA induces apoptosis in testicular cells, possibly through the over-production of ROS. ROS accumulation can reduce mitochondrial membrane potential (MMP) and induced apoptosis (Park et al., 2011). Additional studies have indicated that apoptosis induced by GA is associated with both the intrinsic and extrinsic apoptosis pathways under the regulation of Bcl-2 family proteins. In addition, caspase-9, caspase-8, and caspase-3 were shown to be activated during apoptosis induction by GA (Chen et al., 2014). However, the effects of GA on mammalian reproductive function have not been extensively studied, and the pathway of GA reproductive toxicity has not yet to be determined. In this study, we investigated the mechanisms through which GA impairs the male reproductive function of rat Leydig cells. Thus, the aims of this study were to evaluate the effects of GA on progesterone production and to assess its regulatory effects on ROSdependent apoptosis pathway.

R2C cells were treated with various concentrations of GA for 24 h. The culture media was separated and centrifuged at 400 g for 5 min at 4
C. The culture media supernatants were stored at 20
C until progesterone concentrations were measured using a radioimmunoassay kit (Beijing North Institute of Biological Technology, Beijing, China) according to the manufacturer's instructions (Ge et al., 2007).

2. Materials and methods

2.5. Determination of reactive oxygen species (ROS) generation

2.1. Cell culture and treatments

R2C cells were washed with PBS 3 times, fixed with 100 mL dichlorofluorescin diacetate solution for 30 min at 37
C, and washed 3 times with PBS. Then, 100 mL cell lysis buffer was added to the mixture to stop the reaction. 150 mL of the mixture was transferred to a 96-well culture plate, and then, fluorescence was measured using a fluorometric plate reader at 480 nm/530 nm (Nguyen et al., 2008). Fluorescence was measured once every 30 min for 4 h.

Rat Leydig R2C cells were obtained from the ATCC (Manassas, VA, USA) and cells were cultured in F12 medium (Gibco, Rockville, MD, USA) supplemented with 2.5% FBS (Gibco, Rockville, MD, USA), 15% horse serum (Gibco, Rockville, MD, USA), sodium pyruvate, NaHCO3 and 1% penicillin/streptomycin. The cells were maintained at 37
C with 5% CO2(Sun et al., 2013). For stimulation, cells were treated with 0.5e6 mM GA (J&K Scientific, Beijing, China) for 24 h, and fresh F12 medium was used as the solvent control. The culture media were removed and centrifuged at 400 g for 5 min at 4
C. The supernatants were stored at 20
C for the progesterone assay. 2.2. Cytotoxicity assay and treatments The effects of GA on R2C cell viability were assessed by a MTT assay. Briefly, 4  103 cells per well were seeded in a 96-well flatbottomed plate (Costar, Cambridge, MA, USA); the cells were cultured at 37
C for 24 h and then incubated with different concentrations of GA (0, 0.25, 0.5, 1, 2, 4, and 6 mM) (Manjanatha et al., 2006) for 24 h. Next, 20 mL of MTT (5 mg/mL in PBS) was added to each well, and the cells were incubated at 37
C for 4 h. After the media were removed at the end of the incubation period, 150 mL DMSO (dimethylsulfoxide) was added to each well, and the mixture was shaken at ambient temperature for 10 min. The absorbance was measured at 570 nm using a microplate reader (Thermo Scientific, Chantily, VA, USA). The 25% (IC25), 50% (IC50), and 75% (IC75) maximal inhibitory concentrations of GA in regards to cell number reduction after 24 h of incubation with GA were calculated. Each test was performed in triplicate experiments. 2.3. Single cell gel/comet assay (SCGE) The single cell gel/comet assay was performed as described

2.4. Radioimmunoassay (RIA)

2.6. Measurement of mitochondrial membrane potential (MMP) Disruptions of the mitochondrial electron transport chain, MMP (DJm), and ATP synthesis have been associated with reduced Leydig cell steroidogenesis (Zhang et al., 2011). MMP was measured to evaluate the mitochondrial damage induced by GA in 4 h. The DJm of R2C cells was measured using the fluorescent, lipophilic, and cationic probe JC-1 (Beyotime Biotech, Nantong, China) according to the manufacturer's instructions. After different treatments, cells were incubated with JC-1 staining solution (5 mg/mL) for 20 min at 37
C. Then, the cells were rinsed twice with JC-1 staining buffer and detected by FCM (BD FACS Calibur, San Jose, CA, USA). The ratio of red (i.e., aggregates) to green (i.e., monomers) fluorescence were used to calculate the DJm of R2C cells, and a qualitative assessment of JC-1 uptake by mitochondria was performed for each treatment group. 2.7. Apoptosis analysis The Apoptosis assays were performed by using the Annexin VFITC/PI Apoptosis Detection Kit (Invitrogen, CA, USA). Briefly, after the desired treatments, cells were collected and washed third with binding buffer and then resuspended at a concentration of 1  106 cells/mL in binding buffer. Two hundred microliters of the cell suspension were mixed with 5 mL of Annexin V-FITC and 5 mL of

Please cite this article in press as: Li, M., et al., Glycidamide inhibits progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.09.035

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propidium iodide. After being incubated at room temperature for 10 min, apoptotic cells were determined using a FACScan (BD Biosciences, San Jose, CA, USA). 2.8. Western blot analysis Proteins from the cells were harvested with RIPA lysis buffer (Cell Signaling, Beverly, MA, USA) on ice. Insoluble material was removed from the protein solution by centrifugation at 12,000 g for 15 min at 4
C, and the supernatants were collected. Protein concentration was determined using the BCA assay. The total proteins of the cell samples were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1e2 h at room temperature and then incubated with the appropriate primary antibodies: rabbit anti-rat StAR antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:1000, rabbit anti-rat caspase-3, Bax, and Bcl-2 antibodies (Cell Signal Technology, Beverly, MA, USA) at 1:1000, and rabbit anti-rat GAPDH antibody (Cell Signal Technology, Beverly, MA, USA) at 1:1000. Then, the membranes were incubated with the appropriate secondary antibody. All antibody incubations were performed following standard protocols. The immunoreactive bands were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's protocol.

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dose-response curve, the IC25, IC50, and IC75 of GA were 0.635, 0.872, and 1.198 mM, respectively (Fig. 1A). In cells exposed to three concentrations of GA, the morphology changed from irregular polygons to elliptic at different level. The R2C cells were fragmented, vacuolated and degenerated cytoplasm significantly, especially in the cells with high dose exposure (Fig. 1B). 3.2. Detection of DNA damage in R2C cells after treatment of GA The levels of DNA damage in R2C cells exposed to different doses of GA for 4 h are shown in Fig. 2A. The effects of GA on the DNA of R2C cells were studied using the comet assay and were presented as percent tail DNA, tail length and OTM. Compared with those in the control group, the levels of tail DNA (%), tail length, and OTM increased, and a statistically significant increase in DNA damage was observed at the IC50 and IC75 concentrations of GA (Fig. 2B). 3.3. Effects of GA on progesterone secretion and the expressions of steroidogenic enzymes in R2C cells The dose-dependent progesterone levels of Leydig cells after exposure to GA are shown in Fig. 3. Compared with the negative control, the results showed that the cells treated with different concentrations (IC25, IC50 and IC75) of GA for 24 h presented significantly decreased progesterone production.

2.9. Statistical analysis

3.4. Involvement of ROS in GA-induced R2C cell injury

The reported values are the means of at least three independent replicates and are expressed as the means ± standard deviation (SD). The statistical analysis of significant differences between the control and GA-treated groups was performed using the unpaired student's t-test. Value of p  0.05 (*) were considered to indicate significant differences.

ROS production usually precedes or accompanies the loss of MMP. The intracellular oxidation state of the cells was analyzed by spectrofluorometry with dichlorofluorescin diacetate (DCFHDA). Our data showed that different concentrations (IC25, IC50, and IC75) of GA rapidly increased ROS level at 30 min, and ROS level continued to substantially increase from 1 h to 4 h in R2C cells (Fig. 4A) and became more remarkable at 4 h (Fig. 4B).

3. Results 3.5. Effects of GA on MMP levels 3.1. GA induces injury in R2C cells MTT cassay was conducted to determine the GA concentrations to be used in the following assay. After exposure for 24 h, GA caused a concentration-dependent inhibition of cell viability. Based on the

Mitochondrial membrane potential can cause early apoptosis. To determine the mitochondrial component of the toxic effect of GA in R2C cells, we examined the MMP using JC-1 staining. The DJm of the cells in each treatment group was calculated as the fluorescence

Fig. 1. Determination of inhibitory concentrations of GA on the viability of R2C cells. (A) Cell viability rate was determined by MTT assay after exposure to various concentrations (0e6 mM) of GA. (B) Cell morphology was observed after GA exposure for 24 h R2C cells were treated with different concentrations (IC25, IC50, IC75) of GA (40). The data are presented as the means ± SD of three experiments.

Please cite this article in press as: Li, M., et al., Glycidamide inhibits progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.09.035

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Fig. 2. (A) Representative comet images of control R2C cells and R2C cells incubated with GA. (B) Effects of GA on head DNA (%), tail length, and OTM in R2C cells. Mean ± SD, n ¼ 3. *p < 0.05 vs. control, **p < 0.01 vs. Control.

induce the early apoptosis of R2C cells in a concentrationdependent manner (Fig. 5B). GA activates caspase-3 and regulates Bcl-2 family members to induce apoptosis in R2C cells. We investigated the contribution of caspases to GA-induced apoptosis to determine the signaling pathway responsible for the induction of apoptosis in GA-treated R2C cells. Results of Western blot analysis showed that the protein levels of activated caspase-3 significantly increased in cells exposed to high dose of GA. We also evaluated the expression levels of Bax and Bcl-2 to examine whether the mitochondrial pathway was involved in GA-induced apoptosis. In the R2C cells treated with GA for 24 h, Bax protein levels were increased while Bcl-2 protein levels were gradually decreased in a dose-dependent manner. Thus, the ratio of Bax/Bcl-2 was increased (Fig. 6A and B). 4. Discussions

Fig. 3. R2C cells were treated with different concentrations of GA for 24 h and then progesterone production was determined. Mean ± SD, n ¼ 3. **p < 0.01 vs. control, ***p < 0.001 vs. control.

ratio of second quadrant to fourth quadrant (Fig. 4C). The data of JC1 staining showed that GA induced mitochondrial membrane depolarization (the fluorescence of fourth quadrant), and a significant change was observed as early as 4 h (Fig. 4D). 3.6. Effects of GA on apoptosis levels and the expressions of steroidogenic enzymes and key apoptosis enzymes in R2C cells To investigate the effects of GA on progesterone production, we assessed the changes in the expressions of steroidogenic enzyme genes caused by GA treatment at the protein level. As depicted in Fig. 6A and C, Western blot analysis showed that GA reduced StAR protein levels, suggesting that GA suppresses the expressions of steroidogenic enzymes To determine whether reduction of proliferation capacity of the Leydig cells induced by GA was associated with cells apoptosis, R2C cells were treated with GA as described previously. The ratio of early apoptotic cells (B4:Annexin V þ /PIe) plus late apoptotic cells (B2:Annexin V þ /PI þ) were used to calculate the apoptosis. The apoptosis rate showed an upward trend in B4 and the changes were concentration-dependent (Fig. 5A). The results suggested that GA

GA-associated reproductive toxicity is becoming a popular topic in regards to the daily diet of humans. In the present study, we have examined the underlying mechanisms accounting for GA-induced apoptosis in Leydig cells with he involvement of ROS generation. Our study demonstrated that GA treatment led to the overproduction of ROS, resulting in mitochondrial membrane depolarization. Ultimately, the excess ROS activated the caspase cascade and then apoptotic cell death, and finally resulted in decrease in, progesterone production. Suppression of ROS generation attenuated this inhibition of progesterone production by GA in R2C cells (Fig. 7). The vast majority of androgen is synthesized by and secreted from Leydig cells in the male testes. Functional disorders in Leydig cells profoundly impact the development and maintenance of the male phenotype, the male reproductive tract, and spermatogenesis (Sun et al., 2014). When males transition from the immature phase of life to the adult phase of life, Leydig cells gradually lose their proliferative ability. Therefore, the maintenance of cell numbers and steroidogenic function is essential for Leydig cells, especially with regards to sperm production (Zhang et al., 2011). In the present study, GA inhibited the progesterone production in a dosedependent manner (Fig. 3). In addition, GA induced early apoptosis in Leydig cells and decreased the expression of StAR protein (Fig. 6C). Our data showed that GA treatment at IC75 for 24 h significantly decreased progesterone production by regulating the expression of StAR protein. StAR protein level was detected, because it is a stimulator of steroid hormone biosynthesis. MTT

Please cite this article in press as: Li, M., et al., Glycidamide inhibits progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.09.035

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Fig. 4. Involvement of ROS production and MMP level in GA-induced R2C cell injury. ROS production was determined by fluorescence measurement using the fluorescent probe DCFDA. (A) R2C cells were treated with different concentrations (IC25, IC50, IC75) of GA and exposured to various times (0e180min), then ROS was determined. (B) Effects of different concentrations of GA for 4 h on ROS production of R2C cells. (C) Effects of GA on MMP levels in R2C cells. JC-1 was used to measure fluorescence in control R2C cells(a) and R2C cells incubated with [IC25(b), IC50(c), IC75(d)] of GA for 4 h. (D) The absolute red/green JC-1 intensity ratio was measured (DJm). Mean ± SD, n ¼ 3.*p < 0.05 vs. control, **p < 0.01 vs. control, ***p < 0.001 vs. control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

cytotoxicity assay indicated that, the cell viability decreased with the increase in GA concentration. The single cell gel/comet assay is a well-established genotoxicity test (Musa et al., 2012). It is very useful in in vitro studies, where damage to target cells can be analyzed for the presence of DNA strand breaks. Therefore, in this study, we also examined the possible involvement of DNA damage in the decrease in progesterone production in R2C cells. Several experiments were designed to investigate whether genotoxic mechanisms are involved in the induction of gene expression involved in progesterone production induced by GA in R2C cells. Following the induction of DNA damage, a prominent route of cell inactivation is apoptosis. Studies have showed that, DNA damage could finally induce cell death by activating cell apoptosis (Roos and Kaina, 2013). Early features of cells undergoing apoptosis include cell shrinkage and DNA fragmentation. The SCGE results suggest that GA induced early apoptosis in R2C cells and then affected their progesterone production. Furthermore, a potential mechanism of oxidative damage is the degradation of DNA. Several studies have showed that, ROS could induce DNA damage (Sun et al., 2014). Increasing ROS could

significantly decrease Leydig cell steroidogenesis. Generally, most cells, including R2C cells, could generate ROS during normal energy metabolism. However, the production of toxic oxygen becomes excessive when cells are exposed to chemicals that impair mitochondrial function. ROS could be generated within the mitochondria and damage mitochondrial components (Koizumi et al., 1996). Many studies have showed that, a disproportionate production of ROS in mitochondria led to oxidative stress and dysfunction of cellular organelles, such as mitochondria (Huppertz et al., 2006). This process is associated with the release of mitochondrial factors that trigger the caspase cascade and eventually apoptosis or necrosis (Zhou et al., 2009). Increasing evidence indicates that, ROS play a crucial role in mediating GAinduced cellular apoptosis (Kampfer et al., 2012). Herein we found the significant overproduction of ROS in GA-treated R2C cells (Fig. 4A). Oxidative stress is generally considered an important regulator of cell apoptosis (Bansal et al., 2010). Due o the importance of ROS, the effects of several antioxidants on the regulation of GA-inhibited progesterone production in R2C cells were evaluated. The results

Please cite this article in press as: Li, M., et al., Glycidamide inhibits progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.09.035

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Fig. 5. Effects of GA on apoptosis levels in R2C cells. GA induced apoptosis associated with ROS production. (A) Flow cytometric analysis of cells stained with Annexin V-FITC/PI after treatment with different concentrations [IC25(b), IC50(c), IC75(d)] of GA in 24 h. (B) Statistic results of apoptosis assays. Data are expressed as means ± SD from three independent experiments.*p < 0.05 vs. control, ***p < 0.001 vs. control.

indicated that pretreatment with GSH effectively suppressed the inhibition of progesterone production by GA in R2C cells (the results were not given). The addition of thiol-containing antioxidants, such as GSH, not only eliminated ROS, but also replenished the intracellular stores of endogenous antioxidants (Valko et al., 2007). These results indicate that, GA inside R2C cells could cause an accumulation of ROS, thus induce cell apoptosis, which implies that ROS may serve as an upstream mediator of apoptosis. Mitochondrial respiration is an important intrinsic source of ROS in mammalian cells (Valko et al., 2006). ROS is known to be able to regulate other intracellular signaling cascades, such as mitochondria-mediated cell apoptosis (Bruce-Keller et al., 1998). Steroidogenic cells have developed a strategy to exploit the unique compartmentalization of mitochondrial membranes to regulate steroid synthesis. Studies have showed that, DJm, ATP synthesis, DpH, and [Ca2þ] mt were all required for steroid biosynthesis, and mitochondria were sensitive to oxidative stress (Sun et al., 2013). Cholesterol is transferred into the mitochondria by StAR and cleaved by P450scc during progesterone synthesis, so functional damage to the mitochondria may influence energy metabolism and impair androgen production (Hales et al., 2005). Our study demonstrated that GA significantly reduced the MMP in a dosedependent manner (Fig. 4C and D). Once the MMP are destabilized, the apoptogenic factors, such as cytochrome c, would be released from the outer mitochondria membrane space into the cytosol (Chen et al., 2014). It has been reported that cytochrome c release induces caspase activation (Li et al., 1997). Although we have not detected the release of cytochrome c in our study yet, we would do more in-depth research in the future. GA was found to induce a significant change in MMP earlier than it induced a significant change in progesterone production. In addition, data

showed that StAR protein expression depended on a certain level of

DJm and that perturbation of the mitochondria decreased StAR protein expression. In the present study, GA induced pathologic changes in MMP in R2C cells. Disruption of Djm was thought to be associated with ROS generation (Wang et al., 2012). ROS overproduction may cause lipid, protein, and DNA damage and finally result in cellular apoptosis (Nilsonne et al., 2006). DNA damage can activate many downstream signaling pathways to regulate cellular apoptosis, such as Bcl-2 family proteins and death receptors (Fan et al., 2013). Regardless of the extrinsic or intrinsic apoptotic pathway, caspase-3 is the core protein involved in cellular apoptosis. Activates caspase-3, an executor of apoptosis, which in turn cleaves a number of cellular proteins (Duan et al., 2013). On the other hand, mitochondrial pathway is regulated by Bcl-2 family proteins, such as proapoptotic protein Bax and anti-apoptotic protein Bcl-2 (Pan et al., 2008). The Western blot analysis showed that, the protein level of activated caspase-3 increased and the Bax/Bcl-2 ratio also increased. These results indicate that GA changed the expression levels of apoptosis-related proteins in the treated cells. GA induced early apoptosis in R2C cells after 24 h of treatment. It is possible that ROS overproduction induced by GA diffused into the nucleus and attacked the DNA, inducing DNA-damage apoptosis. This hypothesis was further confirmed by the results of the comet assay. In summary, GA induced early phase apoptosis of mitochondrial pathway and morphological changes in R2C cells. The present data suggest that an excessive intake of GA may lead to severe consequences for male reproduction. In conclusion, we attempted to determine the factors responsible for the effects of GA on progesterone production in R2C cells. Our findings suggest that the reduction of progesterone production

Please cite this article in press as: Li, M., et al., Glycidamide inhibits progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.09.035

Fig. 6. Levels of the StAR and apoptosis-associated proteins expressions determined by western blot analysis. (A) R2C cells were treated with different concentrations of GA for 24 h, the expressions of caspase-3, Bax, Bcl-2 and StAR were determined by western blot, with GAPDH as a loading control. (B) The ratio of caspase-3, Bax and Bcl-2 at the protein level in R2C cells. (C) Effects of GA on the expression levels of StAR protein. Mean ± SD, n ¼ 3. *p < 0.05 vs. control, **p < 0.01 vs. control.

in R2C cells could be caused by GA-induced ROS accumulation. With the increase of ROS, the MMP began to decrease, causing cell apoptosis and inducing decreased secretion of progesterone. In the present study, GA significantly induces apoptosis in R2C cells through a mitochondria-mediated and caspase-dependent pathway. The positive correlation between the overproduction of ROS and mitochondrial dysfunction together with the protective effect of GSH on the GA-induced inhibition of progesterone production suggest that, ROS generation plays a key role in the apoptotic process induced by GA. Therefore, GA could dramatically induce apoptosis in R2C cells by triggering ROS-mediated mitochondria dysfunction and DNA damage, which lead to the inhibition of progesterone production.

Notes The authors declare no competing financial interests.

Funding

Fig. 7. Mechanism of glycidamide inhibiting progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells.

This work is one of the research projects (31471588 and 31201340) supported by National Science Foundation of China (NSFC). The authors also thank Outstanding Young Teachers of the University in Guangdong Province (Yq2013024) and the National Key Technology R&D Program (2012BAK01B03).

Please cite this article in press as: Li, M., et al., Glycidamide inhibits progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.09.035

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Conflicts of interest None. Acknowledgments The authors thank Chen Hong-xia for their help with western blotting, Dai Tao-li for her help with SCGE, and Xu Wei and Sun Wei-wei for their help with the statistical. Xiaoling Li as an associated professor that has just come back to China Mainland from Hong Kong was asked to polish the language in the article. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2016.09.035. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fct.2016.09.035. References Bansal, S., Liu, C.P., Sepuri, N.B., Anandatheerthavarada, H.K., Selvaraj, V., Hoek, J., Milne, G.L., Guengerich, F.P., Avadhani, N.G., 2010. Mitochondria-targeted cytochrome P450 2E1 induces oxidative damage and augments alcohol-mediated oxidative stress. J. Biol. Chem. 285, 24609e24619. Bruce-Keller, A.J., Begley, J.G., Fu, W., Butterfield, D.A., Bredesen, D.E., Hutchins, J.B., Hensley, K., Mattson, M.P., 1998. Bcl-2 protects isolated plasma and mitochondrial membranes against lipid peroxidation induced by hydrogen peroxide and amyloid beta-peptide. J. Neurochem. 70, 31e39. Camacho, L., Latendresse, J.R., Muskhelishvili, L., Patton, R., Bowyer, J.F., Thomas, M., Doerge, D.R., 2012. Effects of acrylamide exposure on serum hormones, gene expression, cell proliferation, and histopathology in male reproductive tissues of Fischer 344 rats. Toxicol. Lett. 211, 135e143. Cengiz, M.F., Gunduz, C.P.B., 2013. Acrylamide exposure among Turkish toddlers from selected cereal-based baby food samples. Food Chem. Toxicol. 60, 514e519. Chen, L., Gong, M.W., Peng, Z.F., Zhou, T., Ying, M.G., Zheng, Q.H., Liu, Q.Y., Zhang, Q.Q., 2014. The marine fungal metabolite, dicitrinone B, induces A375 cell apoptosis through the ROS- related caspase pathway. Mar. Drugs 12, 1939e1958. Chen, W., Feng, L., Shen, Y., Su, H., Li, Y., Zhuang, J., Zhang, L., Zheng, X., 2013. Myricitrin inhibits acrylamide-mediated cytotoxicity in human Caco-2 cells by preventing oxidative stress. Biomed. Res. Int. 2013, 724183. Duan, W.J., Liu, F.L., He, R.R., Yuan, W.L., Li, Y.F., Tsoi, B., Su, W.W., Yao, X.S., Kurihara, H., 2013. Autophagy is involved in the effects of resveratrol on prevention of splenocyte apoptosis caused by oxidative stress in restrained mice. Mol. Nutr. Food Res. 57 (1113), 1145e1157. Fan, C., Chen, J., Wang, Y., Wong, Y.S., Zhang, Y., Zheng, W., Cao, W., Chen, T., 2013. Selenocystine potentiates cancer cell apoptosis induced by 5-fluorouracil by triggering reactive oxygen species-mediated DNA damage and inactivation of the ERK pathway. Free Radic. Biol. Med. 65, 305e316. Friedman, M., 2015. Acrylamide: inhibition of formation in processed food and mitigation of toxicity in cells, animals, and humans. Food Funct. 6, 1752e1772. Ge, R.S., Chen, G.R., Tanrikut, C., Hardy, M.P., 2007. Phthalate ester toxicity in Leydig cells: developmental timing and dosage considerations. Reprod. Toxicol. 23, 366e373. Hales, D.B., Allen, J.A., Shankara, T., Janus, P., Buck, S., Diemer, T., Hales, K.H., 2005. Mitochondrial function in Leydig cell steroidogenesis. Ann. N. Y. Acad. Sci. 1061, 120e134. Hansen, S.H., Olsen, A.K., Soderlund, E.J., Brunborg, G., 2010. In vitro investigations of glycidamide-induced DNA lesions in mouse male germ cells and in mouse and human lymphocytes. Mutat. Res. 696, 55e61. Huppertz, B., Kadyrov, M., Kingdom, J.C., 2006. Apoptosis and its role in the trophoblast. Am. J. Obstet. Gynecol. 195, 29e39. Kampfer, C., Spillner, S., Spinnler, K., Schwarzer, J.U., Terradas, C., Ponzio, R., Puigdomenech, E., Levalle, O., Kohn, F.M., Matzkin, M.E., Calandra, R.S., Frungieri, M.B., Mayerhofer, A., 2012. Evidence for an adaptation in ROS scavenging systems in human testicular peritubular cells from infertility patients. Int. J. Androl. 35, 793e801.

Kim, Y., Ryu, J.C., Choi, H.S., Lee, K., 2011. Effect of 2,20 ,4,40 -tetrahydroxybenzophenone (BP2) on steroidogenesis in testicular Leydig cells. Toxicology 288, 18e26. Koizumi, T., Shirakura, H., Kumagai, H., Tatsumoto, H., Suzuki, K.T., 1996. Mechanism of cadmium-induced cytotoxicity in rat hepatocytes: cadmium-induced active oxygen-related permeability changes of the plasma membrane. Toxicology 114, 125e134. Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S., Wang, X., 1997. Cytochrome c and dATP-dependent formation of Apaf-1/ caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479e489. Manjanatha, M.G., Aidoo, A., Shelton, S.D., Bishop, M.E., McDaniel, L.P., LynCook, L.E., Doerge, D.R., 2006. Genotoxicity of acrylamide and its metabolite glycidamide administered in drinking water to male and female Big Blue mice. Environ. Mol. Mutagen 47, 6e17. Martins, C., Oliveira, N.G., Pingarilho, M., Gamboa da Costa, G., Martins, V., Marques, M.M., Beland, F.A., Churchwell, M.I., Doerge, D.R., Rueff, J., Gaspar, J.F., 2007. Cytogenetic damage induced by acrylamide and glycidamide in mammalian cells: correlation with specific glycidamide-DNA adducts. Toxicol. Sci. 95, 383e390. Mei, N., McDaniel, L.P., Dobrovolsky, V.N., Guo, X., Shaddock, J.G., Mittelstaedt, R.A., Azuma, M., Shelton, S.D., McGarrity, L.J., Doerge, D.R., Heflich, R.H., 2010. The genotoxicity of acrylamide and glycidamide in big blue rats. Toxicol. Sci. 115, 412e421. Musa, M., Kannan, T.P., Masudi, S.M., Rahman, I.A., 2012. Assessment of DNA damage caused by locally produced hydroxyapatite-silica nanocomposite using Comet assay on human lung fibroblast cell line. Mol. Cell. Toxicol. 8, 53e60. Nguyen, T.T., Cho, S.O., Ban, J.Y., Kim, J.Y., Ju, H.S., Koh, S.B., Song, K.S., Seong, Y.H., 2008. Neuroprotective effect of Sanguisorbae radix against oxidative stressinduced brain damage: in vitro and in vivo. Biol. Pharm. Bull. 31, 2028e2035. € m, C., Rundlo €f, A.K., Fernandes, A.P., Bjo €rnstedt, M., Nilsonne, G., Sun, X., Nystro Dobra, K., 2006. Selenite induces apoptosis in sarcomatoid malignant mesothelioma cells through oxidative stress. Free Radic. Biol. Med. 41, 874e885. Pan, M.H., Ghai, G., Ho, C.T., 2008. Food bioactives, apoptosis, and cancer. Mol. Nutr. Food Res. 52, 43e52. Park, S.H., Ozden, O., Jiang, H., Cha, Y.I., Pennington, J.D., Aykin-Burns, N., Spitz, D.R., Gius, D., Kim, H.S., 2011. Sirt3, mitochondrial ROS, ageing, and carcinogenesis. Int. J. Mol. Sci. 12, 6226e6239. Pedreschi, F., Mariotti, M.S., Granby, K., 2014. Current issues in dietary acrylamide: formation, mitigation and risk assessment. J. Sci. Food. Agric. 94, 9e20. Roos, W.P., Kaina, B., 2013. DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis. Cancer Lett. 332, 237e248. Sanderson, J.T., 2006. The steroid hormone biosynthesis pathway as a target for endocrine-disrupting chemicals. Toxicol. Sci. 94 (19), 3e21. Stout, E.P., La Clair, J.J., Snell, T.W., Shearer, T.L., Kubanek, J., 2010. Conservation of progesterone hormone function in invertebrate reproduction. Proc. Natl. Acad. Sci. U. S. A. 107, 11859e11864. Sumner, S.C., Fennell, T.R., Moore, T.A., Chanas, B., Gonzalez, F., Ghanayem, B.I., 1999. Role of cytochrome P450 2E1 in the metabolism of acrylamide and acrylonitrile in mice. Chem. Res. Toxicol. 12, 1110e1116. Sun, J., Bai, S., Bai, W., Zou, F., Zhang, L., Li, G., Hu, Y., Li, M., Yan, R., Su, Z., 2014. 1,3Dichloro-2-propanol inhibits progesterone production through the expression of steroidogenic enzymes and cAMP concentration in Leydig cells. Food Chem. 154, 330e336. Sun, J., Bai, S., Bai, W., Zou, F., Zhang, L., Su, Z., Zhang, Q., Ou, S., Huang, Y., 2013. Toxic mechanisms of 3-monochloropropane-1,2-diol on progesterone production in R2C rat leydig cells. J. Agric. Food Chem. 61, 9955e9960. Valko, M., Leibfritz, D., Moncol, J., Cronin, M.T., Mazur, M., Telser, J., 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39, 44e84. Valko, M., Rhodes, C.J., Moncol, J., Izakovic, M., Mazur, M., 2006. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 160, 1e40. Wang, H., Huang, P., Lie, T., Li, J., Hutz, R.J., Li, K., Shi, F., 2010. Reproductive toxicity of acrylamide-treated male rats. Reprod. Toxicol. 29, 225e230. Wang, Y., Nartiss, Y., Steipe, B., McQuibban, G.A., Kim, P.K., 2012. ROS-induced mitochondrial depolarization initiates PARK2/PARKIN-dependent mitochondrial degradation by autophagy. Autophagy 8, 1462e1476. Wiltbank, M.C., Souza, A.H., Carvalho, P.D., Cunha, A.P., Giordano, J.O., Fricke, P.M., Baez, G.M., Diskin, M.G., 2014. Physiological and practical effects of progesterone on reproduction in dairy cattle. Animal 8 (Suppl. 1), 70e81. Wu, J.J., Wang, K.L., Wang, S.W., Hwang, G.S., Mao, I.F., Chen, M.L., Wang, P.S., 2010. Differential effects of nonylphenol on testosterone secretion in rat Leydig cells. Toxicology 268, 1e7. Zhang, Q., Zou, P., Zhan, H., Zhang, M., Zhang, L., Ge, R.S., Huang, Y., 2011. Dihydrolipoamide dehydrogenase and cAMP are associated with cadmiummediated Leydig cell damage. Toxicol. Lett. 205, 183e189. Zhou, H., Liu, X., Liu, L., Yang, Z., Zhang, S., Tang, M., Tang, Y., Dong, Q., Hu, R., 2009. Oxidative stress and apoptosis of human brain microvascular endothelial cells induced by free fatty acids. J. Int. Med. Res. 37, 1897e1903.

Please cite this article in press as: Li, M., et al., Glycidamide inhibits progesterone production through reactive oxygen species-induced apoptosis in R2C Rat Leydig Cells, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.09.035