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PM2.5 induces male reproductive toxicity via mitochondrial dysfunction, DNA damage and RIPK1 mediated apoptotic signaling pathway
Science of the Total Environment 634 (2018) 1435–1444
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
PM2.5 induces male reproductive toxicity via mitochondrial dysfunction, DNA damage and RIPK1 mediated apoptotic signaling pathway Jin Zhang a,b, Jianhui Liu a,b, Lihua Ren a,c, Jialiu Wei a,b, Junchao Duan a,b, Lefeng Zhang b,d,⁎, Xianqing Zhou a,b,⁎⁎, Zhiwei Sun a,b a
Department of Toxicology and Hygienic Chemistry, School of Public Health, Capital Medical University, Beijing 100069, China Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China School of Nursing, Peking University, Beijing 100191, China d Department of Occupational Health and Environmental Health, School of Public Health, Capital Medical University, Beijing 100069, China b c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• PM2.5 could induce the cell cycle arrest at the G0/G1 phase and proliferation inhibition by ROS induced-DNA damage. • PM2.5 induced cell apoptosis by activating RIPK1 apoptotic signaling pathway, directly. • PM2.5 could destroy mitochondrial structure and cause energy metabolism obstruction, resulting in decrease of sperm motility.
a r t i c l e
i n f o
Article history: Received 14 January 2018 Received in revised form 26 March 2018 Accepted 31 March 2018 Available online xxxx Editor: Jianmin Chen Keywords: PM2.5 DNA damage Apoptosis RIPK1 Mitochondrial damage Male reproductive toxicity
a b s t r a c t Recent years, air pollution has been a serious problem, and PM2.5 is the main air particulate pollutant. Studies have investigated that PM2.5 is a risky factor to the deterioration of semen quality in males. But, the related mechanism is still unclear. To explore the effect of PM2.5, Sprague Dawley (SD) rats were exposed to PM2.5 (0, 1.8, 5.4 and 16.2 mg/kg.bw.) through intratracheal instillation. The exposure was performed once every 3 days and continued for 30 days. In vitro, GC-2spd cells were treated using 0, 50, 100, 200 μg/mL PM2.5 for 24 h. The data showed that sperm relative motility rates and density were remarkably decreased, while sperm malformation rates were significantly increased with exposure to the PM2.5. The expression of Fas/FasL/RIPK1/FADD/ Caspase-8/Caspase-3 and the level of 8-OHdG expression in testes were significantly increased after exposure to PM2.5. Additionally, in vitro the results showed that PM2.5 inhibited cell viability, increased the release of lactate dehydrogenase (LDH) by increasing reactive oxygen species (ROS) level. And ROS induced-DNA damage led to cell cycle arrest at G0/G1 phases and proliferation inhibition. Similar to the vivo study, the expressions of Fas/ FasL/RIPK1/FADD/Caspase-8/Caspase-3 in GC-2spd cells were significantly increased after exposure to PM2.5 for 24-h. In addition, PM2.5 decreased the levels of ATP by impairing mitochondria structures, which led to energy metabolism obstruction resulted in the decrease of sperm motility. The above three aspects together resulted in the decrease in sperm quantity and quality. © 2018 Elsevier B.V. All rights reserved.
⁎ Correspondence to: L. Zhang, Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China. ⁎⁎ Correspondence to: X. Zhou, Department of Toxicology and Hygienic Chemistry, School of Public Health, Capital Medical University, Beijing 100069, China. E-mail addresses: [email protected], (L. Zhang), [email protected]. (X. Zhou).
https://doi.org/10.1016/j.scitotenv.2018.03.383 0048-9697/© 2018 Elsevier B.V. All rights reserved.
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1. Introduction Rapid urbanization and industrialization of China have brought a sharp increase in energy consumption and emissions, and lead to serious air pollution (H. Zhang et al., 2016). Air pollution is a complex process involving different pollutants in the atmosphere. Studies have found that air pollution can damage body's health and the natural environment (Brauer et al., 2012; Kim et al., 2013). On October 17, 2013, the international agency for research on cancer (IARC) announced that the outdoor air pollution was classified as a kind of carcinogen, while atmospheric particulate matter (PM), the main component of outdoor air pollution, was also classified as a carcinogen (Straif et al., 2013). According to the aerodynamic diameter and inhalation capacity through the respiratory tract, PM was divided into three categories (Hasheminassab et al., 2013; Kim et al., 2015), and PM2.5 was considered to be the most representative of air pollutants. PM2.5 pollution has become a serious environmental problem that endangers public health. The formation of haze usually formed through the increase of PM2.5 concentration (Zheng et al., 2015). Reports have indicated that the average annual concentration of PM2.5 in almost all the provincial capitals in China exceeded the national standard of 35 μg/m3 in 2016 (China, 2017), especially for Beijing, the annual PM2.5 pollution is serious (annual PM2.5 mean of 73 μg/m3 in 2016) (Beijing Environmental Protection Bureau, 2017). PM2.5, a complex mixture, could be the carrier of various harmful substances through the strong accumulation effect, such as polycyclic aromatic hydrocarbons (PAH), sulfates, nitrates, ammonium, particlebound water, inorganic ions and organic elemental carbon (Organization, 2013). PM2.5 is easily inhaled and deposited in the bronchi and lungs, reached the alveoli directly. It could enter the bloodstream and transfer to other organs, and cause the disorders (Löndahl et al., 2007). Li et al. found that prolonged exposure of PM2.5 could promote carcinogenesis and metastasis of A549 cell (Li et al., 2016). Also, there was a significant link between the increased exposure of ambient PM2.5 and the development of cardiovascular dysfunction and neurotoxicity (Gorr et al., 2014; Liu et al., 2015), as well as an increased incidence of infertility (Carré et al., 2017; Mahalingaiah et al., 2016). Hammoud and Zhou et al. found that exposure to PM2.5 was a risky factor to the deterioration of semen quality in males (Hammoud et al., 2010; Zhou et al., 2014). Recent studies have proved that PM2.5 could impair blood-testis barrier (BTB) integrity though reducing expressions of tight junction, adherens junction, and gap junction proteins in testicular tissue, followed by oxidative stress (Cao et al., 2017). Our previous study reported that PM2.5 disrupted the blood-testis barrier by activating TGF-β3/p38MAPK pathway (Liu et al., 2018). Meanwhile, epidemiological studies have shown that exposure to outdoor air pollution during pregnancy could also damage the health of pregnant women and fetuses, such as pre-eclampsia (Pedersen et al., 2014), low birth weight (Fleischer et al., 2014), preterm birth, cardiac congenital malformations and intra-uterine growth retardation (Shah and Balkhair, 2011). The previous researches on the reproductive system shows PM2.5 can lead to reproductive toxicity in females and males, as well as developmental toxicity. But, the toxic mechanism is still unclear. Therefore, the combination of SD rats and extracorporeal spermatocyte cells in present study was used to explore the mechanism of the male reproductive toxicity which was caused by PM2.5 from the aspects of mitochondria damage, RIPK1 mediated apoptotic signaling pathway, cell cycle and proliferation. 2. Materials and methods 2.1. Animals and treatments 6–8 week-old Sprague-Dawley male rats were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing,
China). The weight of rats is 180.0–220.0 g, and all of them were fed with the same food and drinking water. The temperature is 22 ± 2 C, the humidity is 60% ± 10% and light cycle is 12-hour light/12-hour dark in the living environment of rats. The animal's use protocol has been approved by Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University, and the ethical review number is AEEI-2016-076. One-week acclimatization later, 32 male rats were randomly divided into control group, low dose PM2.5 group (1.8 mg/kg·bw), moderate dose PM2.5 group (5.4 mg/kg·bw) and high dose PM2.5 group (16.2 mg/kg·bw), 8 rats in each group. According to the annual average concentrations of interim target-1 of PM2.5 (35 μg/m3) recommended by the WHO (Organization, 2007), physiological parameters of 200 g adult rat such as respiratory volume (0.86ML at once breath) and breath rate(85 times per min), as well as a 100-fold uncertainty factor, the low exposure dose of PM2.5 was confirmed to be 1.8 mg/kg bw. Furthermore, 3-fold and 9-fold low-dose was determined as medium exposure dose and high exposure dose of PM2.5, respectively. Rats in PM2.5 groups were treated with varying concentrations PM2.5 (1.8, 5.4, 16.2 mg/kg·bw) through intratracheal instillation, and rats in control group were administered equal volume of physiological saline under the same conditions. Within 30 days, rats were treated once every three days, and then were anesthetized with chloral hydrate (7%, 0.5 mL/100 g·bw) for testes and epididymis on the 30th day after the first dose. The semen of epididymis was collected for detecting sperm parameters, and the testes were stored at −80 °C for subsequent detection. 2.2. PM2.5 collection and extraction The PM2.5 were collected on the roof of a campus building in Fengtai district of Beijing in winter using a large-volume air particle sampler (TH-1000CII, Wuhan Tianhong, China) and quartz fiber filters (8 × 10 in, 47 mm, Pall, USA). The sampling points are set at 6 m above the ground, and the filter samples were equilibrated for 24-h at a constant environment with 25 °C and 30% relative humidity after sampling. Filters were cut into 1 cm2 size, and sonicated 3 times in ultrapure water via an ultrasonator (KQ-250D, Kunshan, Jiangsu, China) in ultrapure water. Then the lotion was collected and filtered with 6-layer gauze. Besides, the mass of extracted particles was weighed and recorded after vacuum freeze drying. Finally, the extracted particles were suspended to a certain concentration of PM2.5 suspension with sterile saline, and shaking with a sonicator (160 W, 20 kHz, 5 min) for 8–10 min before experiments. 2.3. Assessment of sperm parameter in epididymides The epididymis were quickly transferred to 2 mL Dulbecco's Modified Eagle's medium (DMEM, 37 °C) after the rats were anesthetized. The epididymis were cut to release the sperms with a sterile needle, the sperm suspension were detected with an auto system for sperm analysis (Hamilton Thorne IVOS-II,USA), then recorded the results of sperm activity rate and sperm concentration in the computer. Besides, sperm abnormality among 1000 sperms on every slide was counted under an optical microscope (Olympus BX53, Japan) after smear, air drying and hematoxylin–eosin staining. Sperm abnormality = Malformed sperm number ÷ 1000 × 100%. 2.4. Measurement for 8-OHdG 8-Hydroxy-2 deoxyguanosine (8-OHdG), the biomarker of DNA damage in testicular tissue, was measured by using Rat 8-OHdG ELISA Kit (Cusabio Biotech, China). Firstly, 100 mg tissue was homogenized with phosphate buffer saline (PBS) and cell membranes were broken by two freeze-thaw cycles at −20 °C. Then, the supernatant of tissue homogenates was removed to detect the level of 8-OHdG in testicular
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tissue homogenates according to the manufacturer's instruction after 5 min centrifugation. Finally, the levels of 8-OHdG were quantified at 450 nm by using a microplate reader (Themo Multiscan MK3, USA) and curvexpert 1.4.
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standard solution was mixed with 100 μL ATP detection working solution. The luminance was immediately measured using a multifunctional enzyme mark (Enspire, USA). The protein concentration of each treatment group was measured by the protein quantification kit (Dingguo Biotechnology, China).
2.5. Cell culture and experiment design in vitro 2.9. Determination of DNA damage in GC-2spd cells The GC-2spd cell line used in this study was purchased from Guangzhou Jennio Biotech Co., Ltd. (ATCC® number: CRL-2196™). The cells were incubated in Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA) that contained 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin and 100 μg/mL streptomycin, and placed in a temperature incubator that maintained 37 °C and 5% CO2. PM2.5 was dissolved in ultrapure water and used for the experiment after dilution with DMEM (contained 10% FBS). A control group of cells was added to DMEM (contained 10% FBS). 2.6. Detection of cell viability and LDH activity Cell viability of GC-2spd cells was detected via the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were incubated overnight at a density of 3 × 105/mL cells per well in 96-well plates, then exposed to various concentrations of PM2.5 (0, 25, 50, 100, 200, 800 μg/mL) for 24 h. After the 4-hour incubation with 10 μL MTT (5 mg/mL) and 10 min treatment with 150 μl dimethylsulfoxide (DMSO), the optical density of each well were tested at 490 nm through a microplate reader (Thermo Multiskan MK3, USA). Cell viability was presented as the percentage of untreated cells in control group. In addition, LDH detection kit (Jiancheng, China) was used for detection of LDH activity. Firstly, GC-2spd cells in 24-well plates were exposed to PM2.5 for 24 h, and then collected the supernatants and cells to detect the supernatants LDH and total LDH activity according to the manufacturer's protocols. Finally, the absorbance of each group was determined by a microplate reader (Thermo Multiskan MK3, USA) at 492 nm. 2.7. Observation of ultrastructure of GC-2spd cells After 24-h incubation with PM2.5, cells in control (0 μg/mL PM2.5) and 200 μg/mL PM2.5 group were washed with phosphate-buffered saline (PBS) for 2 times. Cells were fixed for 3-h by 2.5% glutaraldehyde at the temperature of 4 °C, and washed three times with 0.1 M Phosphate Buffer (PB). Subsequently, 1% osmium tetroxide solution was used for fixture about 2-h. After another 3-time washing with 0.1 M PB, cells were dehydrated with acetone and a graded series of ethanol, and then dehydrated sample was embedded in epoxy resin. Finally, the embedded sample was cut into ultrathin sections through an ultramicrotome, and a transmission electron microscope (Hitachi HT7700, Japan) was used to observe and photograph cellular ultrastructure. 2.8. Determination of ROS and ATP in GC-2spd cells The level of reactive oxygen species (ROS) in GC-2spd cells was evaluated by using ROS detection kit (Nanjing Jianchen Bioengineering Institute, China). Briefly, GC-2spd cells in 24-well plates exposed to different concentrations PM2.5 (0, 50, 100 and 200 μg/mL) for 24 h, the cells were rinsed two times with PBS. Then cells were marked with 2, 7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) at 37 °C without light for 30 min. Subsequently, cellular sediment was collected after 2time rinsing and centrifugation. After re-suspension with PBS, 10000 cells per tube were collected for fluorescence detection of ROS through flow cytometry (Beckman Coulter, USA). The levels of adenosine triphosphate (ATP) in the cells were measured using ATP assay kit (Biyuntian, China), according to the manufacturer's instructions. After rinsing with PBS, the cells were disrupted in 150 μL lysis buffer, and centrifuged at 12,000 rpm at 4 °C for 5 min, and the supernatant was collected. In 96-well plates, 100 μL sample of the supernatant and ATP
We measured DNA damage using a single-cell gel electrophoresis (Kaiji, Nanjing, China). After being exposed to varying concentrations of PM2.5 for 24-hour, the GC-2spd cells were collected and suspended in PBS. 10 μL cell suspensions and 75 μL low-melting-point agarose preheated to 37 °C were mixed and the suspension was pipetted onto the first gel layer pre-chilled, covered with a piece of clean cover slip at 4 °C for 10 min to develop the second gel layer subsequently, and followed by a third gel layer being adhered to the second one. After being incubated for 30 min at 4 °C in the dark, the slides were immersed in lysate buffer in the dark at 4 °C for 2-h, and then the slides were electrophoresed with 25 V in a gel electrophoresis tank containing fresh alkaline running buffer. After 3 times' washing with PBS, the slides were stained with PI for 10 min in the dark and measured by a fluorescence microscope. The data were analyzed by CASP software based on measurement of 100 randomly scored cells per sample. The percentage of tail DNA was calculated to assess the degree of DNA damage. 2.10. Measurement of cell cycle, proliferation and apoptosis of GC-2spd cells Cell cycle of GC-2spd cells was determined by a cell cycle detection kit (KeyGen, China). After 24-h incubation with PM2.5 (0, 50, 100 and 200 μg/mL), 1 mL cold ethanol (70%) was added to centrifuge tube for cell fixation overnight at 4 °C. After that, cells were rinsed and resuspended with PBS. 100 μL RNase were added to digest intercellular RNA at 37 °C for 30 min, and then intercellular DNA was stained by propidium iodide (400 μL, PI) in the dark for 30 min at 4 °C. The proportion of cells in G0/G1 phase, G2/M phase and S phase were analyzed by using flow cytometry (Beckman Coulter, USA) and MultiCycle V3.0 software. Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) probe diluent (KeyGen, China) were used to mark GC-2spd cells for cell proliferation assay at 37 °C for 15 min. Then cells were well-distributed to the 6-well plates and incubated for 24-h after 2 times washing with PBS. After that, the cells were exposed to PM2.5 for another 24-h. Finally, cells were suspended in PBS and average fluorescence intensity was detected by flow cytometry (Beckman Coulter, USA). Apoptosis of GC2spd cells induced by PM2.5 was measured by using Annexin Vpropidium iodide (PI) apoptosis detection kit (Jiancheng, China). GC2spd cells were incubated with PM2.5 (0, 50, 100 and 200 μg/mL) for 24-h and rinsed with PBS. Then cells were centrifugated at 1200 rpm for 3 min and suspended in binding buffer (500 μL). Subsequently, 5 μL Annexin V-FITC were stained for 15 min as well as 5 μL PI in the dark. N10,000 cells per sample were counted to analyze apoptosis by a flow cytometer (Becton Dickinson, USA). 2.11. Protein expression analysis of relevant factor in RIPK1 apoptotic signaling pathway Protein expressions of Fas, FasL, RIPK1, FADD, Caspase-8, Caspase-3, Cleaved Caspase-8, Cleaved Caspase-3 in testicular tissue and GC-2spd cells were detected via western blot. 30 μg equal amounts of lysate proteins were electrophoresed through the method of SDS polyacrylamide gel electrophoresis (SDS - PAGE), and transferred to nitrocellulose filter (NC) membranes (Millipore, USA) by means of electrical transfer. Then NC membranes were blocked with 5% milk in Tris-buffered saline Tween (TBST) for 1 h at room temperature in the orbital shaker. The membranes were incubated with primary antibodies rabbit-anti- Fas (1: 1000, CST, USA), rabbit-anti- FasL (1: 1000, CST, USA), rabbit-antiRIPK1 (1: 1000, CST, USA), rabbit-anti-FADD (1: 500, Santa Cruz CA,
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Table 1 Effects of PM2.5 on sperm density, motility and abnormal sperm of male rat. Sperm density PM2.5 (mg/kg·bw) (×106/mL)
Sperm motility rate (%)
Sperm abnormality rate (%)
0 1.8 5.4 16.2
75.79 ± 4.38 65.73 ± 5.61 58.53 ± 4.34a 43.12 ± 5.54abc
2.89 ± 0.19 3.46 ± 0.29 3.96 ± 0.18a 6.63 ± 0.24abc
38.02 ± 1.99 31.96 ± 1.87a 27.32 ± 2.06a 18.12 ± 2.08abc
The data are expressed as mean ± S.D. (P b 0.05). a Indicates significant difference compared with the control group. b Indicates significant difference compared with the 5.4 mg/kg PM2.5 group. c Indicates significant difference compared with the 16.2 mg/kg PM2.5group.
USA), rabbit-anti-Caspas8 (1: 1000, CST, USA), rabbit-anti-Caspas3 (1: 1000, CST, USA), rabbit-anti-p-Caspas8 (1: 1000, CST, USA), rabbit-antip-Caspas3 (1: 1000, CST, USA), GAPDH (1: 1000, CST, USA), β-Actin (1:20000, Proteintech, USA) in 4 °C orbital shaker for a night. Furthermore, membranes were incubated with IRDye 800CW goat-anti-rabbit IgG (H + L) in TBS (1:15000; licore, USA) for 1 h in the orbital shaker in the dark at room temperature. After 3 times' washing with TBS, protein bands were visualized on the computer screen through Odyssey CLx Infrared Imaging System (Li-Cor, USA). Finally, Image Lab™ Software (Bio-Rad, USA) was used to analyze the optical density of protein bands. 2.12. Statistical analysis SPSS 17.0 software was used to analyze all the experimental data. Data were analyzed by one-way analysis of variance (ANOVA), followed by Duncan's multiple range test to detect significant differences
between groups. Statistical analysis for comet assay results was assessed using Mann-Whitney rank-sum test. Experimental data were presented in a form of mean ± standard deviation (S.D.), and difference was remarkable at P b 0.05. 3. Results 3.1. Changes of sperm parameters in the epididymis induced by PM2.5 As shown in Table 1, changes of sperm density, motility and abnormal sperm in the epididymis induced by PM2.5 were detected. The sperm density and motility of male rats were significantly decreased in a dose-dependent manner after exposure to PM2.5 for 30 days. The percentage of abnormal had no obvious difference between the control group and 1.8 mg/kg PM2.5 group, while obviously increased in the 5.4 and 16.2 mg/kg PM2.5 groups. 3.2. Changes of protein expression in RIPK1 mediated apoptotic signaling pathway in testes We detected the expressions of Fas, FasL, RIPK1, FADD, Caspase-8 and Caspase-3, which were called relevant factors of RIPK1 apoptotic signaling pathway in testicular tissue. The expression of FAS, FADD and Caspase-3 in the 16.2 mg/kg PM2.5 group was significantly higher than three groups at 0, 1.8 and 5.4 mg/kg (P b 0.05). However, there was no obvious difference in the control group, 1.8 mg/kg PM2.5 group and 5.4 mg/kg PM2.5 group (Fig. 2) (P N 0.05). The expression of FAS-L, RIPK1 and Caspase-8 in testes were significantly increased in a dosedependent manner after exposure to PM2.5 (P b 0.05) (Fig. 1). Results
Fig. 1. Effects of PM2.5 on the protein expressions of RIPK1 apoptotic signaling pathway. (A): The expressions of Fas, FasL, RIPK1, FADD, Caspase8, Caspase3 in testes. (B): Densitometric analysis about these protein bands (Column Diagram). GAPDH was internal control protein. a indicates significant difference compared with the control group, b indicates significant difference compared with the 5.4 mg/kg PM2.5 group, c indicates significant difference compared with the 16.2 mg/kg PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05).
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deposited in the cytoplasm. After the exposure of PM2.5 for 24-h, varisized vacuoles and swollen mitochondria were observed clearly in the cytoplasm, and the mitochondrial ridges were fractured in some cells (Fig. 4B and D).
3.6. Changes of ROS and ATP levels in GC-2spd cells induced by PM2.5
Fig. 2. Expression of 8-OHdG in testes were detected by using ELASA. a indicates significant difference compared with the control group, b indicates significant difference compared with the 5.4 mg/kg PM2.5 group, c indicates significant difference compared with the 16.2 mg/kg PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05).
indicated that the PM2.5 could activate RIPK1 apoptotic signaling pathway in the testicular tissue of rats, and then induced apoptosis. 3.3. Changes of DNA damage in testes induced by PM2.5 The level of 8-OHdG in testes was detected by using Rat 8-OHdG ELISA Kit. The level of 8-OHdG in the 16.2 mg/kg PM2.5 group was significantly higher than three groups at 0, 1.8 and 5.4 mg/kg (P b 0.05). However, there was no obvious difference in the control group, 1.8 mg/kg PM2.5 group and 5.4 mg/kg PM2.5 group (Fig. 2) (P N 0.05).
The ROS level of GC-2spd cells was detected by flow cytometry. As indicated in Fig. 5, the level of ROS in GC-2spd cells was significantly increased in a dose-dependent manner after exposure to PM2.5. Especially, when GC-2spd cells exposed to PM2.5 at dose of 100 and 200 μg/mL, the level of ROS was significantly elevated, and the increase of ROS level was 48% and 85% relative to the control group, respectively (Fig. 5A and B) (P b 0.05). After exposure for 24 h, in the 0, 50 and 100 μg/mL PM2.5 groups, there were no marked change in the level of ATP (P N 0.05). Up to the concentration of 200 μg/mL PM2.5, the levels of ATP significantly decreased compared to that of control, 50 and 100 μg/mL PM2.5 groups respectively (Fig. 5C) (P b 0.05).
3.7. DNA damage of GC-2spd cells Comet assay is a sensitive and intuitionistic method for detecting DNA damage. The percentage of tail DNA in GC-2spd cells was significantly increased in a dose-dependent manner after exposure to PM2.5 for 24-h. Especially, when GC-2spd cells exposed to PM2.5 at dose of 100 and 200 μg/mL, the percentage of tail DNA was significantly elevated relative to the control group and 50 μg/mL PM2.5 group, respectively (Fig. 6) (P b 0.05).
3.4. Cytotoxicity in vitro induced by PM2.5 Cell viability and LDH activity were tested to analyze the adverse effects of PM2.5 on GC-2spd cells. The results of MTT showed that PM2.5 could induce a significant reduction of cell viability when compared to the control, especially when the exposure concentration of PM2.5 was 50, 100, 200, 400 and 800 μg/mL (Fig. 3A) (P b 0.05). Furthermore, the result of LDH was in accordance with cell viability, the ratio of supernatants LDH/Total LDH increased gradually in a dose-dependent (PM2.5) manner (Fig. 3B) (P b 0.05). 3.5. Ultrastructure changes of GC-2spd cells in vitro induced by PM2.5 The cellular ultrastructure in control group and 200 μg/mL PM2.5 group were observed by using transmission electron microscopy (Fig. 4). The mitochondria of GC-2spd cells in the control group showed normal with the shape of oval or short rod, and the mitochondrial crest could be seen clearly (Fig. 4A and C). While the ultrastructure of GC2spd cells in 200 μg/mL PM2.5 group showed that PM2.5 particles were
3.8. Cycle arrest, proliferation and apoptosis of GC-2spd cells induced by PM2.5 The cell cycle, proliferation and apoptosis of GC-2spd cells were analyzed by using flow cytometer. Results of cell cycle analysis showed that the percentage of GC-2spd cells in G0/G1 phase was increased in a dose-dependent manner after 24-treatment of PM2.5 (50, 100, and 200 μg/mL). In this work, there was no statistical difference in S phase relative to the control group, but the cell proportion in G2/M phase significantly decreased when cells exposed to PM2.5 at dose of 200 μg/mL (Fig. 7A) (P b 0.05). Besides, results of cell proliferation showed the average fluorescence intensity increased in a dose-dependent way, and all the experimental groups were significantly higher than the control group (P b 0.05), suggesting that the cell proliferation were inhibited by PM2.5 (Fig. 7B and C). Annexin-V, PI staining and flow cytometric analysis were performed to detect apoptosis of GC-2spd cells. Apoptosis analysis revealed that PM2.5 induced the increase of apoptosis rate in a dose-dependent manner (Fig. 7D and E) (P b 0.05).
Fig. 3. Effects of PM2.5 on the cytotoxicity of GC-2spd cells. (A) Changes of cell viability induced by 24-hour exposure of PM2.5. (B) The ratio of supernatants LDH/Total LDH after 24 h exposure of PM2.5. ⁎ indicates significant difference compared with the control group, # indicates significant difference compared with the 50 μg/mL PM2.5 group, ▲ indicates significant difference compared with the 100 μg/mL PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05). LDH: Lactate dehydrogenase.
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Fig. 4. TEM images of subcellular localization of PM2.5. (A) Control group (2500×), (B) 200 μg/mL PM2.5 group (2500×), (C) Control group (6000×), (D) 200 μg/mL PM2.5 group (6000×). The PM2.5 were internalized into cells and was mainly accumulated in cytoplasm, black arrow points to mitochondria and the white arrow points to PM2.5.
Fig. 5. Effects of PM2.5 on the level of ROS and ATP in GC-2spd cells. (A) Fluorescence intensity of ROS was detected by using Flow Cytometer. (B) Statistical analysis about Fluorescence intensity of ROS. (C) Total ATP levels were expressed as the μmol/g prot. ⁎ indicates significant difference compared with the control group, # indicates significant difference compared with the 50 μg/mL PM2.5 group, ▲ indicates significant difference compared with the 100 μg/mL PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05). ATP: Adenosine triphosphate.
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Fig. 6. DNA damage of GC-2spd cells induced by PM2.5. A: DNA damage of GC-2spd cells induced by various dosages of PM2.5 for 24-h was measured by comet assay and recorded by the fluorescence microscope (400×) (a) Control group, (b) 50 μg/mL PM2.5 group, (c) 100 μg/mL PM2.5 group, (d) 200 μg/mL PM2.5 group. B: Statistical analysis about percentage of tail DNA. ⁎ indicates significant difference compared with the control group, # indicates significant difference compared with the 50 μg/mL PM2.5 group, ▲ indicates significant difference compared with the 100 μg/mL PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05).
3.9. Changes of protein expressions in RIPK1 mediated apoptotic signaling pathway in GC-2spd cells The results showed that the expression of FAS was significantly increased in the 100 and 200 μg/mL PM2.5 treatment groups compared to the control group (P b 0.05); the expression of FAS-L in 200 μg/mL treatment groups was significantly increased compared with the control group, 50 and 100 μg/mL PM2.5 treatment groups (P b 0.05), respectively; the expression of RIPK1, FADD, Cleaved Caspase-8 and Cleaved Caspase-3 in GC-2spd cells were significantly elevated in the 100 and 200 μg/mL PM2.5 treatment groups in a dose-dependent manner (Fig. 8) (P b 0.05). 4. Discussion PM2.5 is the main pollutant in many Chinese cities (Zhang and Cao, 2015). PM2.5 could go deeply into the body through breathing, causing health damage (Peng et al., 2016). Although studies have investigated PM2.5 is a risky factor to the deterioration of semen quality in males, its mechanism is still unclear. In order to investigate the mechanism of reproductive toxicity induced by PM2.5, we firstly demonstrated the effect of PM2.5 on sperm parameters by observing the changes in semen of rats. The results showed that PM2.5 not only reduced sperm density and motility, but also increased the percentage of abnormal sperm in male rats. This is similar to the finding of Hammoud et al., which showed a negative correlation between PM2.5 values and sperm motility, as well as sperm morphology (Hammoud et al., 2010). Furthermore, Radwan et al. reported that exposure to PM2.5 led to an increase of abnormalities in sperm morphology (Radwan et al., 2015). In the present study, the ultrastructure of GC-2spd cells was observed by transmission electron microscope after 24-h exposure to PM2.5. The results showed that PM2.5 could enter the cytoplasm of GC-2spd cells, and varisized vacuoles and swollen mitochondria were observed clearly in the cytoplasm. Gualtieri et al. found that PM2.5 which was collected in winter in Milan could enter A549 cells, and even to induce cell death (Gualtieri et al., 2012). The viability of GC-2spd cells markedly decreased in a dosedependent manner after 24-h exposure to PM2.5 in present study. Besides, the apoptotic rate was in accordance with LDH level that increased significantly after 24-h exposure to PM2.5. Studies have shown that PM2.5 exposure could decrease the cell viability (Gualtieri et al.,
2009), destruct the cell membrane integrity (Alessandria et al., 2014) and result in apoptosis. These results suggested PM2.5 was able to induce cytotoxicity of GC-2spd cells and reproductive injury in male rats. Environmental particulate matter is associated with adverse health outcomes, and many adverse health effects may arise from oxidative stress (Cho et al., 2005). There are plenty of evidences that environmental pollution increases oxidative stress (Filho et al., 2010; Longhin et al., 2013; Zhou et al., 2017). A certain degree of free radicals are necessary for normal sperm function, and excessive free radicals can have deleterious effects on sperm function and subsequent insemination and subsequent health. Evidence has been found that oxidative stress could affect sperm parameters and fertilization (Agarwal and Allamaneni, 2011). The present results showed that ROS level was significantly elevated which suggested that PM2.5 collected in winter in Beijing were able to increase the production of ROS and result in oxidation and anti-oxidative imbalance. ROS could attack DNA, lipids and proteins together with its metabolites, as well as change enzymatic systems, inducing irreversible changes. Research by Gualtieri et al. found that PM2.5 could induce ROS production and damage DNA (Gualtieri et al., 2012). Our results showed that PM2.5 increased the level of 8-OHdG in the high (16.2 mg/kg·bw) dose groups, which was similar to Srám's report that PM2.5 could lead to DNA damage, leading to a decline in fertility (Srám et al., 1999). Growing evidence suggests that the comet assay is a sensitive method for measurement of DNA single-strand breaks at the level of the single cell (Proquin et al., 2017; Wei et al., 2017). The present study showed that PM2.5 can induce high-levels of DNA strand breaks in GC-2spd cells through the comet assay. It has been shown that cell cycle arrest is the initiating biological event in response to DNA damage for repair of broken strands (Jiang et al., 2012). The cell cycle is mainly regulated by cell cycle checkpoint pathways, which would be activated in response to DNA damage (Medema and Macurek, 2012). Our previous study showed that silica nanoparticles induce G0/G1 arrest resulting from DNA damage in GC2spd cells (J. Zhang et al., 2016). So we analyzed cell cycle and cell proliferation by flow cytometry (FCM). The FCM results displayed PM2.5 arrested the cells at G0/G1 phase and inhibited the proliferation of GC2spd cells, which suggested PM2.5 arrest cell cycle and proliferation via ROS induced-DNA damage. As similar to the study of Longhin et al. reported that winter PM2.5 increased the level of ROS and DNA damage (Longhin et al., 2013).
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Fig. 7. Effects of PM2.5 on the cell cycle, proliferation and apoptosis of GC-2spd cells. (A) The proportions of G0/G1, S and G2/M phase cells after exposure to PM2.5. (B, C) Proliferation of HUVECs after PM2.5 treatment was determined by flow cytometry. (D, E) GC-2spd cells treated with PM2.5 showed an increase in the apoptosis rate. ⁎ indicates significant difference compared with the control group, # indicates significant difference compared with the 50 μg/mL PM2.5 group, ▲ indicates significant difference compared with the 100 μg/mL PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05).
However, when the DNA damages are too serious or continue cell cycle arrest, apoptosis would take place (White, 1993). Cheung et al. reported that absence of BRE (DNA damage repair gene), deficiency in DNA damage repair, it would lead to growth arrest at G1 and G2/M phase, and enhance excess apoptosis via Fas/Fas-L signaling pathway in granulosa cells (Yeung et al., 2017). Our previous study has demonstrated that silica nanoparticles could induce apoptosis in spermatogenic cells by activating the RIPK1 pathway, resulting from oxidative stress in male mice (Ren et al., 2016). Wang et al. has found that Fas-L triggers a rapid formation of reactive oxygen species (ROS) in somatic cells, which activates Fas and induces apoptosis (Wang et al., 2008). So we measured expression of apoptotic signal pathway related protein. Fas is an important death receptor on the cell surface, which is activated and transmitted apoptotic signal after the binding of Fas ligand (FasL), inducing the apoptosis. Receptor-Interacting Protein Kinase 1 (RIPK1) is a key kinase in apoptosis and necrotic apoptotic pathways. When Fas is combined with FasL, RIPK1 can be activated and mediated cell apoptosis through caspase-8/caspase3 pathway. RIPK1 also associates
with Fas-associated protein with death domain (FADD), which were required for activation of caspase-8 and apoptosis (Declercq et al., 2009). In present study, the expressions of Fas, Fas-L, RIPK1, FADD, Caspase-8 and Caspase-3 were up-regulated obviously when rats exposed to the high-dose PM2.5 (16.2 mg/kg·bw). And in vitro study, Fas, Fas-L, RIPK1, FADD, Cleaved Caspase-8 and Cleaved Caspase-3 were upregulated, which indicated that the apoptotic signaling pathway were activated by PM2.5 resulting from oxidative stress, leading to cell apoptosis. ROS can induce mitochondrial dysfunction, leading to ATP depletion and ultimately cell death (Burwell and Brookes, 2008). ATP level directly reflects the mitochondrial functional state (Shi et al., 2008). Recent studies reported that PM2.5 inhalation results in mitochondrial damage and reduces ATP production by disrupting the aerobic tricarboxylic acid cycle and oxidative phosphorylation (Gao et al., 2017; Guo et al., 2017). Our previous study has demonstrated the damaged mitochondrial structure could affect cellular energy production, and energy deficiency could affect the maturation process of sperm (Xu et al.,
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Fig. 8. Effects of PM2.5 on the protein expressions of RIPK1 apoptotic signaling pathway. (A): The expressions of Fas, FasL, RIPK1, FADD, Cleaved Caspase-8, Cleaved Caspase-3 in GC-2spd cells. (B): Densitometric analysis about these protein bands (Column Diagram). β-Actin was internal control protein. ⁎ indicates significant difference compared with the control group, # indicates significant difference compared with the 50 μg/mL PM2.5 group, ▲ indicates significant difference compared with the 100 μg/mL PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05).
2014). In our study, the damages of mitochondrial structure in GC-2spd cells were clearly observed in PM2.5-treated group and the level of ATP was significantly decreased after exposure PM2.5. So, energy metabolism dysfunction resulting from mitochondrial damage induced by oxidative stress may be a reason for the decrease of sperm motility rate in this study.
Acknowledgment This study was supported by National Key R&D Program of China (2017YFC0211600, 2017YFC0211606) and National Natural Science Foundation of China (No. 81571130090). Conflict of interest
5. Conclusions On the base of the above experimental results obtained in this study, we made a preliminary conclusion that PM2.5 internalized in the cell could lead to cytotoxicity by inhibiting proliferation and inducing apoptosis, resulting in the decrease in sperm quantity and quality. The toxicological mechanism of PM2.5 involved the following three pathways: (1) PM2.5 could induce the cell cycle arrest at the G0/G1 phase and proliferation inhibition by ROS induced-DNA damage. (2) PM2.5 induced cell apoptosis by activating RIPK1 apoptotic signaling pathway, directly. (3) PM2.5 could destroy mitochondrial structure and cause energy metabolism obstruction, resulting in decrease of sperm motility. The finding may provide some new evidence and clues to understand the mechanism of reproductive toxicity induced by PM2.5. In order to protect public health and the environment, the additional works are deserved for investigating the precise mechanism.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
PM2.5 induces male reproductive toxicity via mitochondrial dysfunction, DNA damage and RIPK1 mediated apoptotic signaling pathway Jin Zhang a,b, Jianhui Liu a,b, Lihua Ren a,c, Jialiu Wei a,b, Junchao Duan a,b, Lefeng Zhang b,d,⁎, Xianqing Zhou a,b,⁎⁎, Zhiwei Sun a,b a
Department of Toxicology and Hygienic Chemistry, School of Public Health, Capital Medical University, Beijing 100069, China Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China School of Nursing, Peking University, Beijing 100191, China d Department of Occupational Health and Environmental Health, School of Public Health, Capital Medical University, Beijing 100069, China b c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• PM2.5 could induce the cell cycle arrest at the G0/G1 phase and proliferation inhibition by ROS induced-DNA damage. • PM2.5 induced cell apoptosis by activating RIPK1 apoptotic signaling pathway, directly. • PM2.5 could destroy mitochondrial structure and cause energy metabolism obstruction, resulting in decrease of sperm motility.
a r t i c l e
i n f o
Article history: Received 14 January 2018 Received in revised form 26 March 2018 Accepted 31 March 2018 Available online xxxx Editor: Jianmin Chen Keywords: PM2.5 DNA damage Apoptosis RIPK1 Mitochondrial damage Male reproductive toxicity
a b s t r a c t Recent years, air pollution has been a serious problem, and PM2.5 is the main air particulate pollutant. Studies have investigated that PM2.5 is a risky factor to the deterioration of semen quality in males. But, the related mechanism is still unclear. To explore the effect of PM2.5, Sprague Dawley (SD) rats were exposed to PM2.5 (0, 1.8, 5.4 and 16.2 mg/kg.bw.) through intratracheal instillation. The exposure was performed once every 3 days and continued for 30 days. In vitro, GC-2spd cells were treated using 0, 50, 100, 200 μg/mL PM2.5 for 24 h. The data showed that sperm relative motility rates and density were remarkably decreased, while sperm malformation rates were significantly increased with exposure to the PM2.5. The expression of Fas/FasL/RIPK1/FADD/ Caspase-8/Caspase-3 and the level of 8-OHdG expression in testes were significantly increased after exposure to PM2.5. Additionally, in vitro the results showed that PM2.5 inhibited cell viability, increased the release of lactate dehydrogenase (LDH) by increasing reactive oxygen species (ROS) level. And ROS induced-DNA damage led to cell cycle arrest at G0/G1 phases and proliferation inhibition. Similar to the vivo study, the expressions of Fas/ FasL/RIPK1/FADD/Caspase-8/Caspase-3 in GC-2spd cells were significantly increased after exposure to PM2.5 for 24-h. In addition, PM2.5 decreased the levels of ATP by impairing mitochondria structures, which led to energy metabolism obstruction resulted in the decrease of sperm motility. The above three aspects together resulted in the decrease in sperm quantity and quality. © 2018 Elsevier B.V. All rights reserved.
⁎ Correspondence to: L. Zhang, Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China. ⁎⁎ Correspondence to: X. Zhou, Department of Toxicology and Hygienic Chemistry, School of Public Health, Capital Medical University, Beijing 100069, China. E-mail addresses: [email protected], (L. Zhang), [email protected]. (X. Zhou).
https://doi.org/10.1016/j.scitotenv.2018.03.383 0048-9697/© 2018 Elsevier B.V. All rights reserved.
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1. Introduction Rapid urbanization and industrialization of China have brought a sharp increase in energy consumption and emissions, and lead to serious air pollution (H. Zhang et al., 2016). Air pollution is a complex process involving different pollutants in the atmosphere. Studies have found that air pollution can damage body's health and the natural environment (Brauer et al., 2012; Kim et al., 2013). On October 17, 2013, the international agency for research on cancer (IARC) announced that the outdoor air pollution was classified as a kind of carcinogen, while atmospheric particulate matter (PM), the main component of outdoor air pollution, was also classified as a carcinogen (Straif et al., 2013). According to the aerodynamic diameter and inhalation capacity through the respiratory tract, PM was divided into three categories (Hasheminassab et al., 2013; Kim et al., 2015), and PM2.5 was considered to be the most representative of air pollutants. PM2.5 pollution has become a serious environmental problem that endangers public health. The formation of haze usually formed through the increase of PM2.5 concentration (Zheng et al., 2015). Reports have indicated that the average annual concentration of PM2.5 in almost all the provincial capitals in China exceeded the national standard of 35 μg/m3 in 2016 (China, 2017), especially for Beijing, the annual PM2.5 pollution is serious (annual PM2.5 mean of 73 μg/m3 in 2016) (Beijing Environmental Protection Bureau, 2017). PM2.5, a complex mixture, could be the carrier of various harmful substances through the strong accumulation effect, such as polycyclic aromatic hydrocarbons (PAH), sulfates, nitrates, ammonium, particlebound water, inorganic ions and organic elemental carbon (Organization, 2013). PM2.5 is easily inhaled and deposited in the bronchi and lungs, reached the alveoli directly. It could enter the bloodstream and transfer to other organs, and cause the disorders (Löndahl et al., 2007). Li et al. found that prolonged exposure of PM2.5 could promote carcinogenesis and metastasis of A549 cell (Li et al., 2016). Also, there was a significant link between the increased exposure of ambient PM2.5 and the development of cardiovascular dysfunction and neurotoxicity (Gorr et al., 2014; Liu et al., 2015), as well as an increased incidence of infertility (Carré et al., 2017; Mahalingaiah et al., 2016). Hammoud and Zhou et al. found that exposure to PM2.5 was a risky factor to the deterioration of semen quality in males (Hammoud et al., 2010; Zhou et al., 2014). Recent studies have proved that PM2.5 could impair blood-testis barrier (BTB) integrity though reducing expressions of tight junction, adherens junction, and gap junction proteins in testicular tissue, followed by oxidative stress (Cao et al., 2017). Our previous study reported that PM2.5 disrupted the blood-testis barrier by activating TGF-β3/p38MAPK pathway (Liu et al., 2018). Meanwhile, epidemiological studies have shown that exposure to outdoor air pollution during pregnancy could also damage the health of pregnant women and fetuses, such as pre-eclampsia (Pedersen et al., 2014), low birth weight (Fleischer et al., 2014), preterm birth, cardiac congenital malformations and intra-uterine growth retardation (Shah and Balkhair, 2011). The previous researches on the reproductive system shows PM2.5 can lead to reproductive toxicity in females and males, as well as developmental toxicity. But, the toxic mechanism is still unclear. Therefore, the combination of SD rats and extracorporeal spermatocyte cells in present study was used to explore the mechanism of the male reproductive toxicity which was caused by PM2.5 from the aspects of mitochondria damage, RIPK1 mediated apoptotic signaling pathway, cell cycle and proliferation. 2. Materials and methods 2.1. Animals and treatments 6–8 week-old Sprague-Dawley male rats were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing,
China). The weight of rats is 180.0–220.0 g, and all of them were fed with the same food and drinking water. The temperature is 22 ± 2 C, the humidity is 60% ± 10% and light cycle is 12-hour light/12-hour dark in the living environment of rats. The animal's use protocol has been approved by Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University, and the ethical review number is AEEI-2016-076. One-week acclimatization later, 32 male rats were randomly divided into control group, low dose PM2.5 group (1.8 mg/kg·bw), moderate dose PM2.5 group (5.4 mg/kg·bw) and high dose PM2.5 group (16.2 mg/kg·bw), 8 rats in each group. According to the annual average concentrations of interim target-1 of PM2.5 (35 μg/m3) recommended by the WHO (Organization, 2007), physiological parameters of 200 g adult rat such as respiratory volume (0.86ML at once breath) and breath rate(85 times per min), as well as a 100-fold uncertainty factor, the low exposure dose of PM2.5 was confirmed to be 1.8 mg/kg bw. Furthermore, 3-fold and 9-fold low-dose was determined as medium exposure dose and high exposure dose of PM2.5, respectively. Rats in PM2.5 groups were treated with varying concentrations PM2.5 (1.8, 5.4, 16.2 mg/kg·bw) through intratracheal instillation, and rats in control group were administered equal volume of physiological saline under the same conditions. Within 30 days, rats were treated once every three days, and then were anesthetized with chloral hydrate (7%, 0.5 mL/100 g·bw) for testes and epididymis on the 30th day after the first dose. The semen of epididymis was collected for detecting sperm parameters, and the testes were stored at −80 °C for subsequent detection. 2.2. PM2.5 collection and extraction The PM2.5 were collected on the roof of a campus building in Fengtai district of Beijing in winter using a large-volume air particle sampler (TH-1000CII, Wuhan Tianhong, China) and quartz fiber filters (8 × 10 in, 47 mm, Pall, USA). The sampling points are set at 6 m above the ground, and the filter samples were equilibrated for 24-h at a constant environment with 25 °C and 30% relative humidity after sampling. Filters were cut into 1 cm2 size, and sonicated 3 times in ultrapure water via an ultrasonator (KQ-250D, Kunshan, Jiangsu, China) in ultrapure water. Then the lotion was collected and filtered with 6-layer gauze. Besides, the mass of extracted particles was weighed and recorded after vacuum freeze drying. Finally, the extracted particles were suspended to a certain concentration of PM2.5 suspension with sterile saline, and shaking with a sonicator (160 W, 20 kHz, 5 min) for 8–10 min before experiments. 2.3. Assessment of sperm parameter in epididymides The epididymis were quickly transferred to 2 mL Dulbecco's Modified Eagle's medium (DMEM, 37 °C) after the rats were anesthetized. The epididymis were cut to release the sperms with a sterile needle, the sperm suspension were detected with an auto system for sperm analysis (Hamilton Thorne IVOS-II,USA), then recorded the results of sperm activity rate and sperm concentration in the computer. Besides, sperm abnormality among 1000 sperms on every slide was counted under an optical microscope (Olympus BX53, Japan) after smear, air drying and hematoxylin–eosin staining. Sperm abnormality = Malformed sperm number ÷ 1000 × 100%. 2.4. Measurement for 8-OHdG 8-Hydroxy-2 deoxyguanosine (8-OHdG), the biomarker of DNA damage in testicular tissue, was measured by using Rat 8-OHdG ELISA Kit (Cusabio Biotech, China). Firstly, 100 mg tissue was homogenized with phosphate buffer saline (PBS) and cell membranes were broken by two freeze-thaw cycles at −20 °C. Then, the supernatant of tissue homogenates was removed to detect the level of 8-OHdG in testicular
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tissue homogenates according to the manufacturer's instruction after 5 min centrifugation. Finally, the levels of 8-OHdG were quantified at 450 nm by using a microplate reader (Themo Multiscan MK3, USA) and curvexpert 1.4.
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standard solution was mixed with 100 μL ATP detection working solution. The luminance was immediately measured using a multifunctional enzyme mark (Enspire, USA). The protein concentration of each treatment group was measured by the protein quantification kit (Dingguo Biotechnology, China).
2.5. Cell culture and experiment design in vitro 2.9. Determination of DNA damage in GC-2spd cells The GC-2spd cell line used in this study was purchased from Guangzhou Jennio Biotech Co., Ltd. (ATCC® number: CRL-2196™). The cells were incubated in Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA) that contained 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin and 100 μg/mL streptomycin, and placed in a temperature incubator that maintained 37 °C and 5% CO2. PM2.5 was dissolved in ultrapure water and used for the experiment after dilution with DMEM (contained 10% FBS). A control group of cells was added to DMEM (contained 10% FBS). 2.6. Detection of cell viability and LDH activity Cell viability of GC-2spd cells was detected via the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were incubated overnight at a density of 3 × 105/mL cells per well in 96-well plates, then exposed to various concentrations of PM2.5 (0, 25, 50, 100, 200, 800 μg/mL) for 24 h. After the 4-hour incubation with 10 μL MTT (5 mg/mL) and 10 min treatment with 150 μl dimethylsulfoxide (DMSO), the optical density of each well were tested at 490 nm through a microplate reader (Thermo Multiskan MK3, USA). Cell viability was presented as the percentage of untreated cells in control group. In addition, LDH detection kit (Jiancheng, China) was used for detection of LDH activity. Firstly, GC-2spd cells in 24-well plates were exposed to PM2.5 for 24 h, and then collected the supernatants and cells to detect the supernatants LDH and total LDH activity according to the manufacturer's protocols. Finally, the absorbance of each group was determined by a microplate reader (Thermo Multiskan MK3, USA) at 492 nm. 2.7. Observation of ultrastructure of GC-2spd cells After 24-h incubation with PM2.5, cells in control (0 μg/mL PM2.5) and 200 μg/mL PM2.5 group were washed with phosphate-buffered saline (PBS) for 2 times. Cells were fixed for 3-h by 2.5% glutaraldehyde at the temperature of 4 °C, and washed three times with 0.1 M Phosphate Buffer (PB). Subsequently, 1% osmium tetroxide solution was used for fixture about 2-h. After another 3-time washing with 0.1 M PB, cells were dehydrated with acetone and a graded series of ethanol, and then dehydrated sample was embedded in epoxy resin. Finally, the embedded sample was cut into ultrathin sections through an ultramicrotome, and a transmission electron microscope (Hitachi HT7700, Japan) was used to observe and photograph cellular ultrastructure. 2.8. Determination of ROS and ATP in GC-2spd cells The level of reactive oxygen species (ROS) in GC-2spd cells was evaluated by using ROS detection kit (Nanjing Jianchen Bioengineering Institute, China). Briefly, GC-2spd cells in 24-well plates exposed to different concentrations PM2.5 (0, 50, 100 and 200 μg/mL) for 24 h, the cells were rinsed two times with PBS. Then cells were marked with 2, 7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) at 37 °C without light for 30 min. Subsequently, cellular sediment was collected after 2time rinsing and centrifugation. After re-suspension with PBS, 10000 cells per tube were collected for fluorescence detection of ROS through flow cytometry (Beckman Coulter, USA). The levels of adenosine triphosphate (ATP) in the cells were measured using ATP assay kit (Biyuntian, China), according to the manufacturer's instructions. After rinsing with PBS, the cells were disrupted in 150 μL lysis buffer, and centrifuged at 12,000 rpm at 4 °C for 5 min, and the supernatant was collected. In 96-well plates, 100 μL sample of the supernatant and ATP
We measured DNA damage using a single-cell gel electrophoresis (Kaiji, Nanjing, China). After being exposed to varying concentrations of PM2.5 for 24-hour, the GC-2spd cells were collected and suspended in PBS. 10 μL cell suspensions and 75 μL low-melting-point agarose preheated to 37 °C were mixed and the suspension was pipetted onto the first gel layer pre-chilled, covered with a piece of clean cover slip at 4 °C for 10 min to develop the second gel layer subsequently, and followed by a third gel layer being adhered to the second one. After being incubated for 30 min at 4 °C in the dark, the slides were immersed in lysate buffer in the dark at 4 °C for 2-h, and then the slides were electrophoresed with 25 V in a gel electrophoresis tank containing fresh alkaline running buffer. After 3 times' washing with PBS, the slides were stained with PI for 10 min in the dark and measured by a fluorescence microscope. The data were analyzed by CASP software based on measurement of 100 randomly scored cells per sample. The percentage of tail DNA was calculated to assess the degree of DNA damage. 2.10. Measurement of cell cycle, proliferation and apoptosis of GC-2spd cells Cell cycle of GC-2spd cells was determined by a cell cycle detection kit (KeyGen, China). After 24-h incubation with PM2.5 (0, 50, 100 and 200 μg/mL), 1 mL cold ethanol (70%) was added to centrifuge tube for cell fixation overnight at 4 °C. After that, cells were rinsed and resuspended with PBS. 100 μL RNase were added to digest intercellular RNA at 37 °C for 30 min, and then intercellular DNA was stained by propidium iodide (400 μL, PI) in the dark for 30 min at 4 °C. The proportion of cells in G0/G1 phase, G2/M phase and S phase were analyzed by using flow cytometry (Beckman Coulter, USA) and MultiCycle V3.0 software. Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) probe diluent (KeyGen, China) were used to mark GC-2spd cells for cell proliferation assay at 37 °C for 15 min. Then cells were well-distributed to the 6-well plates and incubated for 24-h after 2 times washing with PBS. After that, the cells were exposed to PM2.5 for another 24-h. Finally, cells were suspended in PBS and average fluorescence intensity was detected by flow cytometry (Beckman Coulter, USA). Apoptosis of GC2spd cells induced by PM2.5 was measured by using Annexin Vpropidium iodide (PI) apoptosis detection kit (Jiancheng, China). GC2spd cells were incubated with PM2.5 (0, 50, 100 and 200 μg/mL) for 24-h and rinsed with PBS. Then cells were centrifugated at 1200 rpm for 3 min and suspended in binding buffer (500 μL). Subsequently, 5 μL Annexin V-FITC were stained for 15 min as well as 5 μL PI in the dark. N10,000 cells per sample were counted to analyze apoptosis by a flow cytometer (Becton Dickinson, USA). 2.11. Protein expression analysis of relevant factor in RIPK1 apoptotic signaling pathway Protein expressions of Fas, FasL, RIPK1, FADD, Caspase-8, Caspase-3, Cleaved Caspase-8, Cleaved Caspase-3 in testicular tissue and GC-2spd cells were detected via western blot. 30 μg equal amounts of lysate proteins were electrophoresed through the method of SDS polyacrylamide gel electrophoresis (SDS - PAGE), and transferred to nitrocellulose filter (NC) membranes (Millipore, USA) by means of electrical transfer. Then NC membranes were blocked with 5% milk in Tris-buffered saline Tween (TBST) for 1 h at room temperature in the orbital shaker. The membranes were incubated with primary antibodies rabbit-anti- Fas (1: 1000, CST, USA), rabbit-anti- FasL (1: 1000, CST, USA), rabbit-antiRIPK1 (1: 1000, CST, USA), rabbit-anti-FADD (1: 500, Santa Cruz CA,
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Table 1 Effects of PM2.5 on sperm density, motility and abnormal sperm of male rat. Sperm density PM2.5 (mg/kg·bw) (×106/mL)
Sperm motility rate (%)
Sperm abnormality rate (%)
0 1.8 5.4 16.2
75.79 ± 4.38 65.73 ± 5.61 58.53 ± 4.34a 43.12 ± 5.54abc
2.89 ± 0.19 3.46 ± 0.29 3.96 ± 0.18a 6.63 ± 0.24abc
38.02 ± 1.99 31.96 ± 1.87a 27.32 ± 2.06a 18.12 ± 2.08abc
The data are expressed as mean ± S.D. (P b 0.05). a Indicates significant difference compared with the control group. b Indicates significant difference compared with the 5.4 mg/kg PM2.5 group. c Indicates significant difference compared with the 16.2 mg/kg PM2.5group.
USA), rabbit-anti-Caspas8 (1: 1000, CST, USA), rabbit-anti-Caspas3 (1: 1000, CST, USA), rabbit-anti-p-Caspas8 (1: 1000, CST, USA), rabbit-antip-Caspas3 (1: 1000, CST, USA), GAPDH (1: 1000, CST, USA), β-Actin (1:20000, Proteintech, USA) in 4 °C orbital shaker for a night. Furthermore, membranes were incubated with IRDye 800CW goat-anti-rabbit IgG (H + L) in TBS (1:15000; licore, USA) for 1 h in the orbital shaker in the dark at room temperature. After 3 times' washing with TBS, protein bands were visualized on the computer screen through Odyssey CLx Infrared Imaging System (Li-Cor, USA). Finally, Image Lab™ Software (Bio-Rad, USA) was used to analyze the optical density of protein bands. 2.12. Statistical analysis SPSS 17.0 software was used to analyze all the experimental data. Data were analyzed by one-way analysis of variance (ANOVA), followed by Duncan's multiple range test to detect significant differences
between groups. Statistical analysis for comet assay results was assessed using Mann-Whitney rank-sum test. Experimental data were presented in a form of mean ± standard deviation (S.D.), and difference was remarkable at P b 0.05. 3. Results 3.1. Changes of sperm parameters in the epididymis induced by PM2.5 As shown in Table 1, changes of sperm density, motility and abnormal sperm in the epididymis induced by PM2.5 were detected. The sperm density and motility of male rats were significantly decreased in a dose-dependent manner after exposure to PM2.5 for 30 days. The percentage of abnormal had no obvious difference between the control group and 1.8 mg/kg PM2.5 group, while obviously increased in the 5.4 and 16.2 mg/kg PM2.5 groups. 3.2. Changes of protein expression in RIPK1 mediated apoptotic signaling pathway in testes We detected the expressions of Fas, FasL, RIPK1, FADD, Caspase-8 and Caspase-3, which were called relevant factors of RIPK1 apoptotic signaling pathway in testicular tissue. The expression of FAS, FADD and Caspase-3 in the 16.2 mg/kg PM2.5 group was significantly higher than three groups at 0, 1.8 and 5.4 mg/kg (P b 0.05). However, there was no obvious difference in the control group, 1.8 mg/kg PM2.5 group and 5.4 mg/kg PM2.5 group (Fig. 2) (P N 0.05). The expression of FAS-L, RIPK1 and Caspase-8 in testes were significantly increased in a dosedependent manner after exposure to PM2.5 (P b 0.05) (Fig. 1). Results
Fig. 1. Effects of PM2.5 on the protein expressions of RIPK1 apoptotic signaling pathway. (A): The expressions of Fas, FasL, RIPK1, FADD, Caspase8, Caspase3 in testes. (B): Densitometric analysis about these protein bands (Column Diagram). GAPDH was internal control protein. a indicates significant difference compared with the control group, b indicates significant difference compared with the 5.4 mg/kg PM2.5 group, c indicates significant difference compared with the 16.2 mg/kg PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05).
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deposited in the cytoplasm. After the exposure of PM2.5 for 24-h, varisized vacuoles and swollen mitochondria were observed clearly in the cytoplasm, and the mitochondrial ridges were fractured in some cells (Fig. 4B and D).
3.6. Changes of ROS and ATP levels in GC-2spd cells induced by PM2.5
Fig. 2. Expression of 8-OHdG in testes were detected by using ELASA. a indicates significant difference compared with the control group, b indicates significant difference compared with the 5.4 mg/kg PM2.5 group, c indicates significant difference compared with the 16.2 mg/kg PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05).
indicated that the PM2.5 could activate RIPK1 apoptotic signaling pathway in the testicular tissue of rats, and then induced apoptosis. 3.3. Changes of DNA damage in testes induced by PM2.5 The level of 8-OHdG in testes was detected by using Rat 8-OHdG ELISA Kit. The level of 8-OHdG in the 16.2 mg/kg PM2.5 group was significantly higher than three groups at 0, 1.8 and 5.4 mg/kg (P b 0.05). However, there was no obvious difference in the control group, 1.8 mg/kg PM2.5 group and 5.4 mg/kg PM2.5 group (Fig. 2) (P N 0.05).
The ROS level of GC-2spd cells was detected by flow cytometry. As indicated in Fig. 5, the level of ROS in GC-2spd cells was significantly increased in a dose-dependent manner after exposure to PM2.5. Especially, when GC-2spd cells exposed to PM2.5 at dose of 100 and 200 μg/mL, the level of ROS was significantly elevated, and the increase of ROS level was 48% and 85% relative to the control group, respectively (Fig. 5A and B) (P b 0.05). After exposure for 24 h, in the 0, 50 and 100 μg/mL PM2.5 groups, there were no marked change in the level of ATP (P N 0.05). Up to the concentration of 200 μg/mL PM2.5, the levels of ATP significantly decreased compared to that of control, 50 and 100 μg/mL PM2.5 groups respectively (Fig. 5C) (P b 0.05).
3.7. DNA damage of GC-2spd cells Comet assay is a sensitive and intuitionistic method for detecting DNA damage. The percentage of tail DNA in GC-2spd cells was significantly increased in a dose-dependent manner after exposure to PM2.5 for 24-h. Especially, when GC-2spd cells exposed to PM2.5 at dose of 100 and 200 μg/mL, the percentage of tail DNA was significantly elevated relative to the control group and 50 μg/mL PM2.5 group, respectively (Fig. 6) (P b 0.05).
3.4. Cytotoxicity in vitro induced by PM2.5 Cell viability and LDH activity were tested to analyze the adverse effects of PM2.5 on GC-2spd cells. The results of MTT showed that PM2.5 could induce a significant reduction of cell viability when compared to the control, especially when the exposure concentration of PM2.5 was 50, 100, 200, 400 and 800 μg/mL (Fig. 3A) (P b 0.05). Furthermore, the result of LDH was in accordance with cell viability, the ratio of supernatants LDH/Total LDH increased gradually in a dose-dependent (PM2.5) manner (Fig. 3B) (P b 0.05). 3.5. Ultrastructure changes of GC-2spd cells in vitro induced by PM2.5 The cellular ultrastructure in control group and 200 μg/mL PM2.5 group were observed by using transmission electron microscopy (Fig. 4). The mitochondria of GC-2spd cells in the control group showed normal with the shape of oval or short rod, and the mitochondrial crest could be seen clearly (Fig. 4A and C). While the ultrastructure of GC2spd cells in 200 μg/mL PM2.5 group showed that PM2.5 particles were
3.8. Cycle arrest, proliferation and apoptosis of GC-2spd cells induced by PM2.5 The cell cycle, proliferation and apoptosis of GC-2spd cells were analyzed by using flow cytometer. Results of cell cycle analysis showed that the percentage of GC-2spd cells in G0/G1 phase was increased in a dose-dependent manner after 24-treatment of PM2.5 (50, 100, and 200 μg/mL). In this work, there was no statistical difference in S phase relative to the control group, but the cell proportion in G2/M phase significantly decreased when cells exposed to PM2.5 at dose of 200 μg/mL (Fig. 7A) (P b 0.05). Besides, results of cell proliferation showed the average fluorescence intensity increased in a dose-dependent way, and all the experimental groups were significantly higher than the control group (P b 0.05), suggesting that the cell proliferation were inhibited by PM2.5 (Fig. 7B and C). Annexin-V, PI staining and flow cytometric analysis were performed to detect apoptosis of GC-2spd cells. Apoptosis analysis revealed that PM2.5 induced the increase of apoptosis rate in a dose-dependent manner (Fig. 7D and E) (P b 0.05).
Fig. 3. Effects of PM2.5 on the cytotoxicity of GC-2spd cells. (A) Changes of cell viability induced by 24-hour exposure of PM2.5. (B) The ratio of supernatants LDH/Total LDH after 24 h exposure of PM2.5. ⁎ indicates significant difference compared with the control group, # indicates significant difference compared with the 50 μg/mL PM2.5 group, ▲ indicates significant difference compared with the 100 μg/mL PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05). LDH: Lactate dehydrogenase.
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Fig. 4. TEM images of subcellular localization of PM2.5. (A) Control group (2500×), (B) 200 μg/mL PM2.5 group (2500×), (C) Control group (6000×), (D) 200 μg/mL PM2.5 group (6000×). The PM2.5 were internalized into cells and was mainly accumulated in cytoplasm, black arrow points to mitochondria and the white arrow points to PM2.5.
Fig. 5. Effects of PM2.5 on the level of ROS and ATP in GC-2spd cells. (A) Fluorescence intensity of ROS was detected by using Flow Cytometer. (B) Statistical analysis about Fluorescence intensity of ROS. (C) Total ATP levels were expressed as the μmol/g prot. ⁎ indicates significant difference compared with the control group, # indicates significant difference compared with the 50 μg/mL PM2.5 group, ▲ indicates significant difference compared with the 100 μg/mL PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05). ATP: Adenosine triphosphate.
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Fig. 6. DNA damage of GC-2spd cells induced by PM2.5. A: DNA damage of GC-2spd cells induced by various dosages of PM2.5 for 24-h was measured by comet assay and recorded by the fluorescence microscope (400×) (a) Control group, (b) 50 μg/mL PM2.5 group, (c) 100 μg/mL PM2.5 group, (d) 200 μg/mL PM2.5 group. B: Statistical analysis about percentage of tail DNA. ⁎ indicates significant difference compared with the control group, # indicates significant difference compared with the 50 μg/mL PM2.5 group, ▲ indicates significant difference compared with the 100 μg/mL PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05).
3.9. Changes of protein expressions in RIPK1 mediated apoptotic signaling pathway in GC-2spd cells The results showed that the expression of FAS was significantly increased in the 100 and 200 μg/mL PM2.5 treatment groups compared to the control group (P b 0.05); the expression of FAS-L in 200 μg/mL treatment groups was significantly increased compared with the control group, 50 and 100 μg/mL PM2.5 treatment groups (P b 0.05), respectively; the expression of RIPK1, FADD, Cleaved Caspase-8 and Cleaved Caspase-3 in GC-2spd cells were significantly elevated in the 100 and 200 μg/mL PM2.5 treatment groups in a dose-dependent manner (Fig. 8) (P b 0.05). 4. Discussion PM2.5 is the main pollutant in many Chinese cities (Zhang and Cao, 2015). PM2.5 could go deeply into the body through breathing, causing health damage (Peng et al., 2016). Although studies have investigated PM2.5 is a risky factor to the deterioration of semen quality in males, its mechanism is still unclear. In order to investigate the mechanism of reproductive toxicity induced by PM2.5, we firstly demonstrated the effect of PM2.5 on sperm parameters by observing the changes in semen of rats. The results showed that PM2.5 not only reduced sperm density and motility, but also increased the percentage of abnormal sperm in male rats. This is similar to the finding of Hammoud et al., which showed a negative correlation between PM2.5 values and sperm motility, as well as sperm morphology (Hammoud et al., 2010). Furthermore, Radwan et al. reported that exposure to PM2.5 led to an increase of abnormalities in sperm morphology (Radwan et al., 2015). In the present study, the ultrastructure of GC-2spd cells was observed by transmission electron microscope after 24-h exposure to PM2.5. The results showed that PM2.5 could enter the cytoplasm of GC-2spd cells, and varisized vacuoles and swollen mitochondria were observed clearly in the cytoplasm. Gualtieri et al. found that PM2.5 which was collected in winter in Milan could enter A549 cells, and even to induce cell death (Gualtieri et al., 2012). The viability of GC-2spd cells markedly decreased in a dosedependent manner after 24-h exposure to PM2.5 in present study. Besides, the apoptotic rate was in accordance with LDH level that increased significantly after 24-h exposure to PM2.5. Studies have shown that PM2.5 exposure could decrease the cell viability (Gualtieri et al.,
2009), destruct the cell membrane integrity (Alessandria et al., 2014) and result in apoptosis. These results suggested PM2.5 was able to induce cytotoxicity of GC-2spd cells and reproductive injury in male rats. Environmental particulate matter is associated with adverse health outcomes, and many adverse health effects may arise from oxidative stress (Cho et al., 2005). There are plenty of evidences that environmental pollution increases oxidative stress (Filho et al., 2010; Longhin et al., 2013; Zhou et al., 2017). A certain degree of free radicals are necessary for normal sperm function, and excessive free radicals can have deleterious effects on sperm function and subsequent insemination and subsequent health. Evidence has been found that oxidative stress could affect sperm parameters and fertilization (Agarwal and Allamaneni, 2011). The present results showed that ROS level was significantly elevated which suggested that PM2.5 collected in winter in Beijing were able to increase the production of ROS and result in oxidation and anti-oxidative imbalance. ROS could attack DNA, lipids and proteins together with its metabolites, as well as change enzymatic systems, inducing irreversible changes. Research by Gualtieri et al. found that PM2.5 could induce ROS production and damage DNA (Gualtieri et al., 2012). Our results showed that PM2.5 increased the level of 8-OHdG in the high (16.2 mg/kg·bw) dose groups, which was similar to Srám's report that PM2.5 could lead to DNA damage, leading to a decline in fertility (Srám et al., 1999). Growing evidence suggests that the comet assay is a sensitive method for measurement of DNA single-strand breaks at the level of the single cell (Proquin et al., 2017; Wei et al., 2017). The present study showed that PM2.5 can induce high-levels of DNA strand breaks in GC-2spd cells through the comet assay. It has been shown that cell cycle arrest is the initiating biological event in response to DNA damage for repair of broken strands (Jiang et al., 2012). The cell cycle is mainly regulated by cell cycle checkpoint pathways, which would be activated in response to DNA damage (Medema and Macurek, 2012). Our previous study showed that silica nanoparticles induce G0/G1 arrest resulting from DNA damage in GC2spd cells (J. Zhang et al., 2016). So we analyzed cell cycle and cell proliferation by flow cytometry (FCM). The FCM results displayed PM2.5 arrested the cells at G0/G1 phase and inhibited the proliferation of GC2spd cells, which suggested PM2.5 arrest cell cycle and proliferation via ROS induced-DNA damage. As similar to the study of Longhin et al. reported that winter PM2.5 increased the level of ROS and DNA damage (Longhin et al., 2013).
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Fig. 7. Effects of PM2.5 on the cell cycle, proliferation and apoptosis of GC-2spd cells. (A) The proportions of G0/G1, S and G2/M phase cells after exposure to PM2.5. (B, C) Proliferation of HUVECs after PM2.5 treatment was determined by flow cytometry. (D, E) GC-2spd cells treated with PM2.5 showed an increase in the apoptosis rate. ⁎ indicates significant difference compared with the control group, # indicates significant difference compared with the 50 μg/mL PM2.5 group, ▲ indicates significant difference compared with the 100 μg/mL PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05).
However, when the DNA damages are too serious or continue cell cycle arrest, apoptosis would take place (White, 1993). Cheung et al. reported that absence of BRE (DNA damage repair gene), deficiency in DNA damage repair, it would lead to growth arrest at G1 and G2/M phase, and enhance excess apoptosis via Fas/Fas-L signaling pathway in granulosa cells (Yeung et al., 2017). Our previous study has demonstrated that silica nanoparticles could induce apoptosis in spermatogenic cells by activating the RIPK1 pathway, resulting from oxidative stress in male mice (Ren et al., 2016). Wang et al. has found that Fas-L triggers a rapid formation of reactive oxygen species (ROS) in somatic cells, which activates Fas and induces apoptosis (Wang et al., 2008). So we measured expression of apoptotic signal pathway related protein. Fas is an important death receptor on the cell surface, which is activated and transmitted apoptotic signal after the binding of Fas ligand (FasL), inducing the apoptosis. Receptor-Interacting Protein Kinase 1 (RIPK1) is a key kinase in apoptosis and necrotic apoptotic pathways. When Fas is combined with FasL, RIPK1 can be activated and mediated cell apoptosis through caspase-8/caspase3 pathway. RIPK1 also associates
with Fas-associated protein with death domain (FADD), which were required for activation of caspase-8 and apoptosis (Declercq et al., 2009). In present study, the expressions of Fas, Fas-L, RIPK1, FADD, Caspase-8 and Caspase-3 were up-regulated obviously when rats exposed to the high-dose PM2.5 (16.2 mg/kg·bw). And in vitro study, Fas, Fas-L, RIPK1, FADD, Cleaved Caspase-8 and Cleaved Caspase-3 were upregulated, which indicated that the apoptotic signaling pathway were activated by PM2.5 resulting from oxidative stress, leading to cell apoptosis. ROS can induce mitochondrial dysfunction, leading to ATP depletion and ultimately cell death (Burwell and Brookes, 2008). ATP level directly reflects the mitochondrial functional state (Shi et al., 2008). Recent studies reported that PM2.5 inhalation results in mitochondrial damage and reduces ATP production by disrupting the aerobic tricarboxylic acid cycle and oxidative phosphorylation (Gao et al., 2017; Guo et al., 2017). Our previous study has demonstrated the damaged mitochondrial structure could affect cellular energy production, and energy deficiency could affect the maturation process of sperm (Xu et al.,
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Fig. 8. Effects of PM2.5 on the protein expressions of RIPK1 apoptotic signaling pathway. (A): The expressions of Fas, FasL, RIPK1, FADD, Cleaved Caspase-8, Cleaved Caspase-3 in GC-2spd cells. (B): Densitometric analysis about these protein bands (Column Diagram). β-Actin was internal control protein. ⁎ indicates significant difference compared with the control group, # indicates significant difference compared with the 50 μg/mL PM2.5 group, ▲ indicates significant difference compared with the 100 μg/mL PM2.5 group. The data are expressed as mean ± S.D. (P b 0.05).
2014). In our study, the damages of mitochondrial structure in GC-2spd cells were clearly observed in PM2.5-treated group and the level of ATP was significantly decreased after exposure PM2.5. So, energy metabolism dysfunction resulting from mitochondrial damage induced by oxidative stress may be a reason for the decrease of sperm motility rate in this study.
Acknowledgment This study was supported by National Key R&D Program of China (2017YFC0211600, 2017YFC0211606) and National Natural Science Foundation of China (No. 81571130090). Conflict of interest
5. Conclusions On the base of the above experimental results obtained in this study, we made a preliminary conclusion that PM2.5 internalized in the cell could lead to cytotoxicity by inhibiting proliferation and inducing apoptosis, resulting in the decrease in sperm quantity and quality. The toxicological mechanism of PM2.5 involved the following three pathways: (1) PM2.5 could induce the cell cycle arrest at the G0/G1 phase and proliferation inhibition by ROS induced-DNA damage. (2) PM2.5 induced cell apoptosis by activating RIPK1 apoptotic signaling pathway, directly. (3) PM2.5 could destroy mitochondrial structure and cause energy metabolism obstruction, resulting in decrease of sperm motility. The finding may provide some new evidence and clues to understand the mechanism of reproductive toxicity induced by PM2.5. In order to protect public health and the environment, the additional works are deserved for investigating the precise mechanism.
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