The apoptosis in arsenic-induced oxidative stress is associated with autophagy in the testis tissues of chicken

The apoptosis in arsenic-induced oxidative stress is associated with autophagy in the testis... ABSTRACT The aim of this study is to investigate whether arsenic (As) could induce testicular poisoning and influence the oxidative stress, apoptosis and autophagy in chickens. Seventy-two 1-day-old male Hy-line chickens were divided into 4 groups which were exposed to 0, 0.625, 1.25, and 2.5 mg/kg body weight (BW) of arsenic trioxide (As2O3) for 30, 60 and 90 days, respectively. Histological and ultrastructural changes, antioxidant enzyme activity, mRNA and protein levels of apoptosis and autophagy-related genes were detected. Oxidative stress injuries were obvious in the testes exposure to As2O3, which resulted in the decreased activities of antioxidant enzymes, such as catalase (CAT) and superoxide dismutases (SOD). Meanwhile, the changes of mRNA and protein levels of apoptosis and autophagy-related genes showed that As2O3 exposure induced enhanced testicular apoptosis and increased the levels of autophagy markers such as Microtubule associated protein light chains 3-II (LC3-II), dynein, Beclin-1, Autophagy associated gene 5 (ATG5) and ATG4B but not LC3-I and mammalian target of rapamycin (mTOR), and demonstrated the cross-talk between apoptosis and autophagy. Histological and ultrastructural abnormalities confirm the changes of the above indicators. Taken together, our findings provide deeper insights into roles of excessive apoptosis and autophagy in the aggravation of testicular damage, which could contribute to a better understanding of As2O3-induced testicular poisoning in chickens. INTRODUCTION The increasing use of heavy metals and metalloids in human activities has raised concerns about possible health risks. The health hazard caused by heavy metals and metalloids poisoning depends on the type and concentration of metals, sorts and contact points, which are composed of acute and chronic toxicity (Rathi et al., 2017). Arsenic (As), which has been studied for centuries, is abundant in the earth's crust, soil, water, almost all tissues of animals and plants and is an important topic in both mainstream media and the scientific literature (Martinez et al., 2011). Arsenic is classified as a class Ι carcinogen by the international agency for research on cancer (IARC) and specified the critical value of drinking water as 10 μg/L by the World Health Organization (WHO) and the U.S. Environmental Protection Agency (EPA) (Brandon et al., 2014). However, excessive levels of As are present in many countries, which have a harmful impact on the environment. For example, As content above the threshold has led to acute toxicological consequences in the West Bengal region of India as well as Bangladesh. Similar situations are also reported in Poland, Pakistan, Serbia, Hungary, Iran, and Basque Country in Spain (Hettick et al., 2015). Zhao et al. (2017b) have proved that exposure to arsenic trioxide (A2O3) can cause neurotoxicity in chickens. Dietary A2O3 can trigger apoptosis in immune organs of chickens (Zhao et al., 2017a). Further research on As exposure is necessary due to persistent exposure to humans and animals. Oxidative stress is part of the main mechanisms of inorganic arsenic (iAs) poisoning (Gao et al., 2013). Sodium arsenite (NaAsO2) exposure toxicity can stimulate reactive oxygen species (ROS) and cause oxidative stress by affecting the balance between the pro-oxidant and the antioxidant in the body (Samuel et al., 2005). In the defense mechanisms against oxidative stress, the free radical scavenger glutathione (GSH) and antioxidant enzymes such as catalase (CAT), superoxide dismutases (SOD) and glutathione peroxidase (GPx) are one of the most important defense mechanisms of ROS, which are good indicators of assessment to evaluate the A2O3 toxicity (White et al., 2003). However, lipid peroxidation occurs when these enzymes are not able to offset excess ROS (Mena et al., 2009). Malondialdehyde (MDA) is an end product of lipid peroxidation, mainly comes from peroxidation of polyunsaturated fatty acids in biological system, is widely used as a biological marker to evaluate oxidative damage (Zhao et al., 2017b). Apoptosis and autophagy are two major pathways of programmed cell death under developmental and/or stressful conditions, and they are closely related (Bongaerts, 2008). Apoptosis can be triggered via the extrinsic pathway, which is activated through Tumor Necrosis Factor (TNF) and Factor associated suicide (Fas), or the intrinsic pathway, which is mediated by B cell lymphoma/leukemia 2 (Bcl-2) gene family releasing. Moreover, both pathways induce apoptosis through activation of effector caspases (Yang et al., 2017). Autophagy is an evolutionary mechanism in all eukaryotic cells that plays a key role in maintaining cell homeostasis and adapting to various stress conditions, and mediates by various proteins like Beclin-1 and Microtubuleassociated protein light chains 3 (LC3) (Cherra et al., 2010). The cross-talk between apoptosis and autophagy can exist at the level of signal transduction pathways that sense death/survival signals and translate them into molecular events that activate or repress specific death processes (Eisenberg-Lerner et al., 2009). Recent studies have demonstrated that Bcl-2 gene family which is well-known as apoptosis-related genes can also regulate autophagy. An apoptosis-related gene, Bcl-2, also showed inhibition of autophagy. For example, Beclin-1 can bind Bcl-2/Bcl-XL to form constitutive Bcl-2/Bcl-XL-Beclin-1 to reduce Beclin-1 monomeric to inhibit autophagy (Levine et al., 2008). Moreover, protein 53 (p53) can regulate autophagy through inhibiting the mammalian target of rapamycin (mTOR) pathway (Feng et al., 2005). mTOR is considered as a key homeostatic regulator by upregulated protein, lipid synthesis and inhibiting excessive autophagy. What's more, mTOR not only regulates autophagy, but is also regulated by apoptosis factors, which further illustrates the cross-talk between autophagy and apoptosis (Corradetti and Guan, 2006). In addition, in the case of oxidative stress, excessive ROS can activate both apoptosis and autophagy. Kroemer et al. (2010) showed that autophagy can be activated by ROS in cardiomyocytes. Studies have proved that lead (Pb) can affect the balance of mitochondrial dynamics by inducing oxidative stress, resulting in autophagy, which ultimately causes the chicken spleen immune dysfunction (Han et al., 2017). As2O3 could trigger extrinsic and intrinsic apoptosis pathways in immune organs of chickens, meanwhile, ROS generated by oxidative stress might be an important driver of excessive apoptosis (Zhao et al., 2017a). Manganese (Mn) induced oxidative stress and subsequent DNA damage and apoptosis in testicular tissues of chickens (Liu et al., 2013b). Whereas, adverse effects of As2O3 exposure to testicular tissues of chickens have not been explored before. Therefore, it is necessary to research the mechanism of testes exposure to As2O3 and the cross-talk between apoptosis and autophagy in this study. Testes are important reproductive organs of chickens, which are related to the production and reproduction of the poultry industry. If the testicles cause poisoning, the quality of sperm in the testes will be affected, eventually leading to sperm dysplasia or deformity, which will affect the success rate of mating. Or even if the mating is successful, the offspring will be stunted, which will affect the meat quality and harm the development of the breeding industry. In addition, As is now a ubiquitous poisonous metalloid and cases of excessive As have been reported repeatedly. So it makes more sense to study the effects of As on the testicles of chickens. We have previously studied the effects of As2O3 poisoning on testicular inflammation (Sun et al., 2017b), in this experiment, we use the histopathology, Transmission electron microscopy (TEM) and TdT-mediated dUTP nick end labeling (TUNEL) assay to observe the ultrastructural and apoptosis index of the testes, detect the CAT, GSH, MDA and ability of anti-hydroxy radical (AHR) levels to analyze oxidative stress, utilize Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot to assay the expressions of autophagy- and apoptosis-related genes. Reveal the effects of A2O3 on testicular poisoning in chickens, including disordered antioxidant system, increased apoptosis and autophagy, the cross-talk between apoptosis and autophagy, and slightly tissues damage. MATERIALS AND METHOD Animals and Treatment All procedures used in the present study were approved by the Institutional Animal Care and Use Committee of Northeast Forestry University in Harbin, China (approval no. UT-31; 20 June 2014). Seventy-two 1-day-old male Hy-line chickens, (purchased by Weiwei Co. Ltd. Harbin, China) were divided into four groups at random: the control group (C group), the low-As2O3 group (L group), the middle-As2O3 group (M group) and the high-As2O3 group (H group) (18 chickens/group). Throughout the experimental period, feed and water were allowed ad libitum. Feeding, housing and handling of experimental animals were conducted according to the established commercial managemental practices. The basal diet was formulated according to the National Research Council (NRC) (1994). The composition of the basal diet for chickens was 2.766% metabolizable energy (kcal/kg), 18.45% crude protein, 2.82% crude fiber, 3.80% calcium, and 0.62% phosphorus. Treatment groups were maintained on a low-As2O3-sufficient diet (L group) containing 0.625 mg/kg body weight (BW), corresponding 7.5 mg/kg feed, a middle-As2O3-sufficient diet (M group) containing 1.25 mg/kg BW, 15 mg/kg feed, and a high-As2O3-sufficient diet (H group) containing 2.5 mg/kg BW, 30 mg/kg feed. The highest dose of sub-chronic toxicity test can use 1/20 to 1/5 of the median lethal dose [Supplementary Appendix 1], and the median lethal dose of As for chicken was 50 mg/kg BW [Supplementary Appendix 2]. As2O3 was purchased from the New Technology Development Company, CHINA AGR UNIV. Six chickens of each group were selected randomly and euthanized by sodium pentobarbital after stress termination on days 30, 60 and 90 of the experiment, respectively. Then, the testes were quickly excised and rinsed with ice-cold 0.9% NaCl solution and frozen immediately in liquid nitrogen, and stored at −80°C until required (Liu et al., 2013a). Histological Observations The samples of testes were prepared as follows: dehydrating, clearing, embedding, baking. Finally, the sections (5 mm) were stained with hematoxylin and eosin and observed. Transmission Electron Microscopy The testis tissues (1 mm3) were rapidly extracted from the typical sample in all H groups. TEM was performed as described previously (Liu et al., 2016). TUNEL Assay Testis tissues processing for apoptosis-related DNA fragmentation were evaluated by TUNEL Assay using the In situ Cell Death Detection kit (Roche Diagnostics, Shanghai, China) according to the manufacturer's instruction. Results were expressed as the percentage of TUNEL-positive cells among the total number of cells counted (Yao et al., 2013a). Determination of Antioxidant System CAT, GSH, MDA, SOD, GPx, total antioxidant capacity (T-AOC) and ability of AHR levels in testis tissues (n = 5/group) indices indicated in the figures were detected using detection kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's protocol. Determination of the mRNA Expression of Autophagy- and Apoptosis-related Genes by RT-qPCR Total RNA was isolated from testis tissue samples (50 mg tissue; n = 5/group) were isolated from synchronized populations by using Trizol reagent (Takara, Japan). cDNA was created by the PrimeScriptTM RT Reagent Kit (Takara, Japan) and used for qPCR analysis, afterwards. Specific primers used for amplification were shown in Supplementary Appendix 3. The details were indicated in previous research (Li et al., 2017). Table 1. Body weights and testicular weights after As2O3 exposure.   Body weight (g)  Testis weight (g)  Testis weight/Body weight (%)  C group  1600 ± 14.7  4.40 ± 0.10  0.275  L group  1600 ± 16.1  2.03 ± 0.16*  0.127*  M group  1560 ± 20.0  1.51 ± 0.05*  0.097*  H group  1580 ± 15.9  1.14 ± 0.12**  0.072**    Body weight (g)  Testis weight (g)  Testis weight/Body weight (%)  C group  1600 ± 14.7  4.40 ± 0.10  0.275  L group  1600 ± 16.1  2.03 ± 0.16*  0.127*  M group  1560 ± 20.0  1.51 ± 0.05*  0.097*  H group  1580 ± 15.9  1.14 ± 0.12**  0.072**  Values represent the mean S.D. *P < 0.05, **P < 0.01, n = 5. View Large Table 1. Body weights and testicular weights after As2O3 exposure.   Body weight (g)  Testis weight (g)  Testis weight/Body weight (%)  C group  1600 ± 14.7  4.40 ± 0.10  0.275  L group  1600 ± 16.1  2.03 ± 0.16*  0.127*  M group  1560 ± 20.0  1.51 ± 0.05*  0.097*  H group  1580 ± 15.9  1.14 ± 0.12**  0.072**    Body weight (g)  Testis weight (g)  Testis weight/Body weight (%)  C group  1600 ± 14.7  4.40 ± 0.10  0.275  L group  1600 ± 16.1  2.03 ± 0.16*  0.127*  M group  1560 ± 20.0  1.51 ± 0.05*  0.097*  H group  1580 ± 15.9  1.14 ± 0.12**  0.072**  Values represent the mean S.D. *P < 0.05, **P < 0.01, n = 5. View Large Western Blot Analysis of Autophagy- and Apoptosis-related Genes Protein extracts were prepared by using SDS Lysis Buffer, protein concentration was quantified using Enhanced BCA Protein Assay Kit (Beyotime, China) according to the manufacturer's protocol. Proteins were separated by electrophoresis through 15% SDS-polyacrylamide gels and transferred to PVDF membranes (Yao et al., 2013b). The membranes were incubated with diluted primary antibodies against LC3 I/II (1:1000, Wanleibio, China), dynein (1:5000, Abcam, USA), Beclin-1 (1:1000, Novusbio, USA), Autophagy associated gene 5 (ATG5)/ATG4B (1:500, ABclonal, China), TORC1/Cytochrome c (Cyt c) (1:500, Bioss, China), Bcl-2/Caspase-9/Bcl-2 Associated X Protein (Bax)/p53/Caspase-3 (1:1000, Wanleibio, China), Caspase-8/anti -β-actin monoclonal antibody (1:2000, proteintech, China). Subsequently, horseradish peroxidase-conjugated secondary antibody were used to detect specific reaction products. The details were indicated in previous research (Zhao et al., 2017a). Statistical Analysis The experimental data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 5.0 software. SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was utilized for all the statistical analyses conducted in this study. If there were significant differences between treated groups and the control group, a one way ANOVA was used to assess them. Differences between means were assessed using Tukey's paired test between the diverse treated groups. P values less than 0.05 were considered to indicate statistically significant. RESULTS Testis and Body Weights As shown in Table 1, on 90 days post-treatment, body weight was similar between chickens in all groups. The testes of As2O3 treated animals were significantly smaller than those of the control chickens (P < 0.05, P < 0.01), reflecting the generalized toxicity of this compound. The ratios of testis weight to body weight decreased (P < 0.05, P < 0.01) in all treatment groups, compared to the control group. Histological Analysis The results of histological observations of testes on day 90 are shown in Figure 1. Compared to the C group (Figure 1a), part of spermatogenic cells including spermatogonium, primary spermatocyte, secondary spermatocyte and spermatid in the seminiferous tubules were vacuolar and degenerative in L, M and H groups (Figure 1b–d). Figure 1. View largeDownload slide Histopathological changes in the testis tissues of chickens (×400). a. C group. b. L group. c. M group. d. H group on 90 day. Blue, green, yellow and red arrows in C group indicate spermatogonium, primary spermatocyte, secondary spermatocyte and spermatid, respectively. And in L, M and H groups, the same colored arrows represent the corresponding diseased cells, respectively. Figure 1. View largeDownload slide Histopathological changes in the testis tissues of chickens (×400). a. C group. b. L group. c. M group. d. H group on 90 day. Blue, green, yellow and red arrows in C group indicate spermatogonium, primary spermatocyte, secondary spermatocyte and spermatid, respectively. And in L, M and H groups, the same colored arrows represent the corresponding diseased cells, respectively. Ultrastructural Analysis As shown in Figure 2, some representative pictures of testis tissues samples on days 30, 60 and 90 of the experiment in H group illustrated the As-induced ultrastructural changes in testis tissues. Transmission electron microscopy revealed normal testicular ultrastructure in C group (Figure 2a). In stark contrast, As2O3 exposure caused serious abnormalities in testis tissues. Typical characteristics of apoptosis, such as condensation and margination of nuclear chromatin were also found, as well as swollen mitochondria with the degeneration or loss of cristae. In addition, numerous autophagosomes-like structures were obviously presented in the testes of As2O3-treated groups, compared to the C group (Figure 2b–d). Figure 2. View largeDownload slide Developmental exposure to As induced ultrastructural abnormalities in chicken testes. Representative transmission electron microscopy images of testicular tissues from a. C group. b. H group on 30 day, c. 60 day and d. 90 day. Double arrows: typical characteristics of apoptosis; fold arrows: swollen mitochondria; thin arrows: autophagosomes, n = 5. Figure 2. View largeDownload slide Developmental exposure to As induced ultrastructural abnormalities in chicken testes. Representative transmission electron microscopy images of testicular tissues from a. C group. b. H group on 30 day, c. 60 day and d. 90 day. Double arrows: typical characteristics of apoptosis; fold arrows: swollen mitochondria; thin arrows: autophagosomes, n = 5. TUNEL Assay The results of TUNEL assay were showed in Figure 3. Normal cell apoptosis appeared in the testis tissues of the C group (Figure 3a). But the number of apoptosis cells was significantly increased in H groups on days 30, 60 and 90, compared with corresponding C group (P < 0.05, P < 0.01) (Figure 3b–e). Figure 3. View largeDownload slide Representative images of TUNEL stained testicular cross sections from a. C group. b. H group on 30 day, c. 60 day and d. 90 day (×400). e. Apoptosis index. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Figure 3. View largeDownload slide Representative images of TUNEL stained testicular cross sections from a. C group. b. H group on 30 day, c. 60 day and d. 90 day (×400). e. Apoptosis index. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Results of Analysis of Oxidative Stress and Antioxidant Indices CAT, GSH, MDA, SOD, GPx, T-AOC and ability of AHR levels in the chicken testes on days 30, 60 and 90 are presented in Figure 4. In general, the activities of CAT significantly decreased (P < 0.05) in all treatment groups on days 30, 60 and 90, expect the L group on day 30 (P > 0.05) (Figure 4a). The activities of GSH declined remarkably (P < 0.05) in all treatment groups on days 60 and 90, and decreased in H group on day 30, however, had no significant change in L and M group (P > 0.05) (Figure 4b). The GPx levels were decreased in all treatment groups on days 60 and 90 (P < 0.05, P < 0.01), however, it had no significant change on day 30 (Figure 4g). T-AOC, SOD and the ability of AHR were decreased in all treatment groups (P < 0.05, P < 0.01), compared to the corresponding C group (Figure 4c, e, f). Whereas, the concentrations of MDA content were significantly increased in all treatment groups on day 90 and in H group on days 30 and 60 (P < 0.05) (Figure 4d). Figure 4. View largeDownload slide Results of analysis of oxidative stress and antioxidant indices in the testis tissues of chickens. a. Represented the results of CAT. b. Represented the results of GSH. c. Represented the results of AHR. d. Represented the results of MDA contents. e. Represented the results of T-AOC. f. Represented the results of SOD. g. Represented the results of GPx. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Figure 4. View largeDownload slide Results of analysis of oxidative stress and antioxidant indices in the testis tissues of chickens. a. Represented the results of CAT. b. Represented the results of GSH. c. Represented the results of AHR. d. Represented the results of MDA contents. e. Represented the results of T-AOC. f. Represented the results of SOD. g. Represented the results of GPx. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Effects of As2O3 on Apoptosis-related Genes’ mRNA and Protein Levels in the Testis Tissues of Chickens Effects of As2O3 treatment on the levels of the apoptosis-related genes’ mRNA and proteins (p53, Bcl-2, Bax, Cyt c, Caspase-9, Caspase-3, Caspase-8) in the chicken testes were showed in Figure 5. Compared with the C group, the treatment of As2O3 in H group was significantly upregulated the expressions of all the detected apoptosis-related genes’ mRNA and protein (P < 0.05, P < 0.01), except the expression of Bcl-2. The mRNA and protein levels of Bcl-2 were downregulated markedly, compared to the corresponding C group (Figure 5a–c) (P < 0.05). Figure 5. View largeDownload slide Effects of apoptosis-related genes’ mRNA and protein levels in the testis tissues of chickens. a. Represented the mRNA levels of apoptosis-related genes in the testes. b and c Represented the protein levels of apoptosis-related genes. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Figure 5. View largeDownload slide Effects of apoptosis-related genes’ mRNA and protein levels in the testis tissues of chickens. a. Represented the mRNA levels of apoptosis-related genes in the testes. b and c Represented the protein levels of apoptosis-related genes. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Effects of As2O3 on Autophagy-related Genes’ mRNA and Protein Levels in the Testis Tissues of Chickens Effects of As2O3 treatment on the levels of the autophagy-related genes’ mRNA and proteins in the chicken testes were showed in Figure 6. Compared with the C group, the mRNA and protein levels of ATG5, ATG4B, Beclin-1, dynein and LC3 II/I presented higher levels in H group (P < 0.05, P < 0.01), in a dose-dependent manner. However, the mRNA and protein levels of TORC1 were markedly decreased, compared to the corresponding C group (Figure 6a–c) (P < 0.05, P < 0.01). Figure 6. View largeDownload slide Effects of autophagy-related genes’ mRNA and protein levels in the testis tissues of chickens. a. Represented the mRNA levels of autophagy-related genes in the testes. b and c. Represented the protein levels of autophagy-related genes. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Figure 6. View largeDownload slide Effects of autophagy-related genes’ mRNA and protein levels in the testis tissues of chickens. a. Represented the mRNA levels of autophagy-related genes in the testes. b and c. Represented the protein levels of autophagy-related genes. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. DISCUSSION Arsenic is considered as the most harmful substance in the Agency for Toxic Substances and Disease Registry (Zhao et al., 2017b). Numerous reports have shown that chronic As2O3 exposure can cause the damages of the organisms, i.e., skins, lungs and bladder cancers, cardiovascular dysfunction, adverse pregnancy outcomes, cognitive deficits and type-2 diabetes (Abdul et al., 2015). Previous researches have suggested that As2O3 induced testicular toxicity that can cause inflammatory and heat shock responses in chickens (Sun et al., 2017b). In this research, we aim to analyze the correlation between the exposure to As2O3 and testicular ultrastructural changes, oxidative stress, apoptosis and autophagy, as well as the cross-talk between apoptosis and autophagy after As2O3 exposure. Testis weight, the ratios of testis weight, testicular histopathology and TUNEL assays (Table 1, Figures 2 and 3) were utilized to appraise the extent of cell death resulting from As2O3 treatment; showing vacuolar and degenerative spermatogenic cells and apoptotic cells in the treatment groups which meant the degeneration and necrosis of cells and the decline of the testicular function. Ultrastructural analysis and TUNEL assay have intuitively demonstrated that As2O3 exposure to testes can induce apoptosis and autophagy (Figures 2 and 3) in a time-dependent manner. Since oxidative stress can induce apoptosis and autophagy, we have conducted a correlation analysis of oxidative damage. Antioxidant enzyme activities in the testes of chickens varied with As2O3 concentrations and duration of As2O3 exposure. Likewise, a significantly dose-dependent decrease of GSH was found during the exposure. In chickens, the activities of antioxidant enzymes, such as GPx and CAT, were inhibited after As2O3 exposure depending on exposure time and concentration in immune organs and brain tissues (Zhao et al., 2017a; Zhao et al., 2017b). CAT has the effect of catalyzing the decomposition of highly toxic hydrogen peroxide (H2O2) into water and molecular oxygen (Adeyemi et al., 2015). SOD removes superoxide radicals by converting them into H2O2, which is rapidly converted into water by CAT. SOD and CAT work together to eliminate ROS, and the SOD-CAT system forms the first line of defence against oxidative stress (Wafa et al., 2011). The major function of GPx is reducing soluble H2O2 and converting it into water in the presence of oxidated glutathione (Sun et al., 2016). In the present study, the CAT, SOD and GPx activities were reduced after As2O3 exposure, which means the reduced detoxification capacity. GSH is known to play a role in protecting cells from ROS damage through metal toxicity (Hellou et al., 2012). In this study, the level of GSH is depleted after As2O3 exposure, which increased the susceptibility of the testes to free radical-induced toxicity. Simultaneously, the ability of AHR is down-regulated in a dose-dependent fashion. What's more, the level of T-AOC decreased, which was more indicative of the decline in the testicular antioxidant capacity. In addition, MDA is a product of lipid peroxidation, which is widely used to assess oxidative damage in various environmental attacks (Wang et al., 2008). MDA content increased in chickens brain tissues exposure to As2O3 (Zhao et al., 2017b). According to previous studies, with the increase of dietary As content, the content of MDA gradually increased in this study, indicating that the lipid peroxidation and greater extent of oxidative damage were enhanced in As2O3-exposed models. Previous reports indicate that oxidative stress induced by NaAsO2 may lead to apoptosis and necrosis in rat proximal tubular cells (Prabu and Muthumani, 2012). As2O3 can induce oxidative stress, and cause autophagy, finally lead to the neurotoxicity in chicken brain tissues (Sun et al., 2017a). However, in chickens, it is not well clear whether the As2O3-induced testes undergo apoptosis and/or autophagy. Accordingly the current study was designed to understand the mechanistic approach adopted by testes in response to As2O3-induced in chicken, and to observe the cross-talk between apoptosis and autophagy. Apoptosis is a programmed cell death in which the specific signaling pathways (extrinsic and intrinsic apoptosis pathways) are activated. It is well known that important apoptosis regulatory mechanism includes death receptors, caspases, mitochondria, Bcl-2 and tumor-suppressor genes are well known as important apoptosis regulatory mechanisms (Alamolhodaei et al., 2015). Apoptosis induced by NaAsO2 depends on exposure time and concentration (Chen et al., 1998). In this study, As2O3-induced apoptosis was mediated through the increases of caspases activation, p53 and the corresponding changes of Bcl-2 gene family. It is reported that oxidative stress may trigger apoptosis (Xie et al., 2017). Oxidative stress accumulates too much •OH that cannot be cleared by the body later they cause DNA damage. What's more, DNA damage can increase the level of p53 which can regulate gene expression of Bcl-2 gene family (Yu et al., 2001). A pro-apoptotic member of the Bcl-2 gene family called Bax increased significantly in a dose-dependent fashion. Meanwhile, Bcl-2, the anti-apoptotic member was declined after As2O3 exposure in the current study. Increased Bax further stimulated Cyt c, thereby activated Caspase-9 up-regulation, and finally generated Caspase-3, which led to apoptosis (Eisenberg-Lerner et al., 2009). This is a pathway to cause apoptosis called the intrinsic pathway. In this study, after As2O3 exposure, intrinsic pathway of apoptosis-related genes’ mRNA and protein levels such as Cyt c, p53, Caspase-9 Caspase-3 and Bax were increased as a dose dependent fashion, compared to the corresponding C group, which meant intrinsic pathway of apoptosis was activated by As2O3 exposure. In addition, the extrinsic pathway of apoptosis was also activated. In the present study, Fas is activated after oxidative stress, which can be associated with caspase activation, such as Caspase-8 and Caspase-3 (Wajant, 2002). Caspase-3 is a key effector molecule for apoptosis execution. Most factors that trigger apoptosis eventually require Caspase-3-mediated signal transduction pathways, leading to apoptosis (Rong et al., 2008). Our study demonstrated that As2O3 significantly increased Fas, Caspase-8 and Caspase-3 expressions in a dose-dependent fashion, which means extrinsic pathway of apoptosis is activated. What's more, we found that numerous factors (Cyt c, p53, Bax, Bcl-2, Caspase-9) controlled intrinsic apoptosis, which should be more notable in this study. Autophagy is a cellular degradation pathway which used to remove damaged or redundant proteins and organelles. It is often thought of as a natural process for maintaining cellular homeostasis and the reaction of cells to the progress of starvation, infection or disease (Chen et al., 2015). The study has pointed out that atrazine and chlorpyrifos and their combination can induce oxidative stress and autophagy in the immune organs of common carp (Chen et al., 2015). Accumulating evidence indicates that Beclin-1 participates in the early stages of autophagosome formation, facilitating the nucleation of autophagosomes (Kihara et al., 2001). Our research revealed that the mRNA and the protein levels of Beclin-1 were increased. Cao et al. (2009) showed that the mRNA of Beclin-1 is increased significantly in rats after pilocarpine-induced status epilepticus. Our results show that Beclin-1 has similar changes in the level of stress response in birds as in mammals. LC3 and dynein are widely used to monitor autophagy. Under the stimulus of autophagy, LC3 converts its soluble cytoplasmic form (LC3-I) into the form of autophagy associated with membrane expansion (LC3-II). The ratio of LC3-II/LC3-I was used to evaluate the number of autophagosomes (Qu et al., 2015). Our results showed that after As2O3 exposure, the mRNA and protein levels of the ratio of LC3-II/LC3-I up-regulated, meanwhile, the mRNA and protein levels of dynein were increased significantly, compared to the corresponding C group, supporting the presence of enhanced autophagy in chickens testes exposed to As2O3. Besides, ATG4B and ATG5 are also involved in the formation of autophagosomes, which contact Beclin-1 with LC3 and dynein (Eisenberg-Lerner et al., 2009). This research showed that the levels of ATG4B and ATG5 were both up-regulated in all As2O3-treated groups, compared to the corresponding C group. Eisenberg-Lerner et al. (2009) manifested that apoptosis and autophagy are not only mutually antagonistic pathways, but also show a synergistic effect and also offset each other. Therefore, we explore the cross-talk between apoptosis and autophagy in this study. mTOR plays a crucial positive moderating role in cell growth, proliferation, cell cycle and so on. The function of mTOR is primarily mediated by mTORC1 and mTORC2. The occurrence of autophagy is mainly regulated by mTORC1, therefore, we explore the changes in TORC1 after As2O3 exposure (Wei et al., 2012). Autophagy activity was negatively regulated by mTOR. In current study, the levels of TORC1 declined remarkably in all As2O3-treated groups, compared to the corresponding C group, which meant autophagy was activated. What's more, there are studies show that mTOR can stimulate the expression of Beclin-1, simultaneously, Beclin-1 is a main regulator of autophagy, and several proteins such as Bcl-2 family members interact with it (Qu et al., 2015). That's already mentioned that Beclin-1 can bind Bcl-2/Bcl-XL to form constitutive Bcl-2/Bcl-XL-Beclin-1 to reduce Beclin-1 monomeric to inhibit autophagy. However, in current study, the levels of Beclin-1 were highly increased, and Bcl-2 levels were inhibited in a dose-dependent fashion, which meant Beclin-1 monomeric increased and apoptosis and autophagy were both activated. Besides, the inhibition of mTOR is also regulated by p53. In current study, the levels of p53 were markedly increased in a dose-dependent fashion, which also suppressed the level of mTOR and promoted autophagy. To sum up, Bcl-2 genes family and p53, which are apoptosis-related genes, can adjust autophagy by regulating the level of mTOR, which in turn acts on apoptosis-related genes to regulate apoptosis. In a word, apoptosis and autophagy can promote each other in testis tissues of chickens after As2O3 exposure. CONCLUSION To sum up, the findings in the present study provide some preliminary evidences that exposure to As2O3 can cause tissue damage in testes of chickens, which will affect the breeding of the poultry industry and people's economic benefits. We have showed that As2O3 exposure led to ultrastructural changes, oxidative stress, apoptosis and autophagy in the testes of chickens. Arsenic-induced oxidative stress can cause the cross-talk between apoptosis and autophagy in testes of chicken. Acknowledgements This study was supported by the National Natural Science Foundation of China (Grant No. 31672619); the Fundamental Research Funds for the Central Universities (Grant No. 2572016EAJ5) and the Natural Science Foundation of Heilongjiang Province (Grant No. C2015061). Notes The appropriate scientific section for the paper: Physiology and Reproduction. REFERENCES Abdul K. S., Jayasinghe S. S., Chandana E. P., Jayasumana C., De Silva P. M.. 2015. Arsenic and human health effects: A review. Chemosphere . 40: 238– 245. Adeyemi J. A., Da C. M. A., Barbosa F. J.. 2015. Teratogenicity, genotoxicity and oxidative stress in zebrafish embryos (Danio rerio) co-exposed to arsenic and atrazine. Comp. Biochem. Physiol. C Toxicol. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

The apoptosis in arsenic-induced oxidative stress is associated with autophagy in the testis tissues of chicken

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© 2018 Poultry Science Association Inc.
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0032-5791
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Abstract

ABSTRACT The aim of this study is to investigate whether arsenic (As) could induce testicular poisoning and influence the oxidative stress, apoptosis and autophagy in chickens. Seventy-two 1-day-old male Hy-line chickens were divided into 4 groups which were exposed to 0, 0.625, 1.25, and 2.5 mg/kg body weight (BW) of arsenic trioxide (As2O3) for 30, 60 and 90 days, respectively. Histological and ultrastructural changes, antioxidant enzyme activity, mRNA and protein levels of apoptosis and autophagy-related genes were detected. Oxidative stress injuries were obvious in the testes exposure to As2O3, which resulted in the decreased activities of antioxidant enzymes, such as catalase (CAT) and superoxide dismutases (SOD). Meanwhile, the changes of mRNA and protein levels of apoptosis and autophagy-related genes showed that As2O3 exposure induced enhanced testicular apoptosis and increased the levels of autophagy markers such as Microtubule associated protein light chains 3-II (LC3-II), dynein, Beclin-1, Autophagy associated gene 5 (ATG5) and ATG4B but not LC3-I and mammalian target of rapamycin (mTOR), and demonstrated the cross-talk between apoptosis and autophagy. Histological and ultrastructural abnormalities confirm the changes of the above indicators. Taken together, our findings provide deeper insights into roles of excessive apoptosis and autophagy in the aggravation of testicular damage, which could contribute to a better understanding of As2O3-induced testicular poisoning in chickens. INTRODUCTION The increasing use of heavy metals and metalloids in human activities has raised concerns about possible health risks. The health hazard caused by heavy metals and metalloids poisoning depends on the type and concentration of metals, sorts and contact points, which are composed of acute and chronic toxicity (Rathi et al., 2017). Arsenic (As), which has been studied for centuries, is abundant in the earth's crust, soil, water, almost all tissues of animals and plants and is an important topic in both mainstream media and the scientific literature (Martinez et al., 2011). Arsenic is classified as a class Ι carcinogen by the international agency for research on cancer (IARC) and specified the critical value of drinking water as 10 μg/L by the World Health Organization (WHO) and the U.S. Environmental Protection Agency (EPA) (Brandon et al., 2014). However, excessive levels of As are present in many countries, which have a harmful impact on the environment. For example, As content above the threshold has led to acute toxicological consequences in the West Bengal region of India as well as Bangladesh. Similar situations are also reported in Poland, Pakistan, Serbia, Hungary, Iran, and Basque Country in Spain (Hettick et al., 2015). Zhao et al. (2017b) have proved that exposure to arsenic trioxide (A2O3) can cause neurotoxicity in chickens. Dietary A2O3 can trigger apoptosis in immune organs of chickens (Zhao et al., 2017a). Further research on As exposure is necessary due to persistent exposure to humans and animals. Oxidative stress is part of the main mechanisms of inorganic arsenic (iAs) poisoning (Gao et al., 2013). Sodium arsenite (NaAsO2) exposure toxicity can stimulate reactive oxygen species (ROS) and cause oxidative stress by affecting the balance between the pro-oxidant and the antioxidant in the body (Samuel et al., 2005). In the defense mechanisms against oxidative stress, the free radical scavenger glutathione (GSH) and antioxidant enzymes such as catalase (CAT), superoxide dismutases (SOD) and glutathione peroxidase (GPx) are one of the most important defense mechanisms of ROS, which are good indicators of assessment to evaluate the A2O3 toxicity (White et al., 2003). However, lipid peroxidation occurs when these enzymes are not able to offset excess ROS (Mena et al., 2009). Malondialdehyde (MDA) is an end product of lipid peroxidation, mainly comes from peroxidation of polyunsaturated fatty acids in biological system, is widely used as a biological marker to evaluate oxidative damage (Zhao et al., 2017b). Apoptosis and autophagy are two major pathways of programmed cell death under developmental and/or stressful conditions, and they are closely related (Bongaerts, 2008). Apoptosis can be triggered via the extrinsic pathway, which is activated through Tumor Necrosis Factor (TNF) and Factor associated suicide (Fas), or the intrinsic pathway, which is mediated by B cell lymphoma/leukemia 2 (Bcl-2) gene family releasing. Moreover, both pathways induce apoptosis through activation of effector caspases (Yang et al., 2017). Autophagy is an evolutionary mechanism in all eukaryotic cells that plays a key role in maintaining cell homeostasis and adapting to various stress conditions, and mediates by various proteins like Beclin-1 and Microtubuleassociated protein light chains 3 (LC3) (Cherra et al., 2010). The cross-talk between apoptosis and autophagy can exist at the level of signal transduction pathways that sense death/survival signals and translate them into molecular events that activate or repress specific death processes (Eisenberg-Lerner et al., 2009). Recent studies have demonstrated that Bcl-2 gene family which is well-known as apoptosis-related genes can also regulate autophagy. An apoptosis-related gene, Bcl-2, also showed inhibition of autophagy. For example, Beclin-1 can bind Bcl-2/Bcl-XL to form constitutive Bcl-2/Bcl-XL-Beclin-1 to reduce Beclin-1 monomeric to inhibit autophagy (Levine et al., 2008). Moreover, protein 53 (p53) can regulate autophagy through inhibiting the mammalian target of rapamycin (mTOR) pathway (Feng et al., 2005). mTOR is considered as a key homeostatic regulator by upregulated protein, lipid synthesis and inhibiting excessive autophagy. What's more, mTOR not only regulates autophagy, but is also regulated by apoptosis factors, which further illustrates the cross-talk between autophagy and apoptosis (Corradetti and Guan, 2006). In addition, in the case of oxidative stress, excessive ROS can activate both apoptosis and autophagy. Kroemer et al. (2010) showed that autophagy can be activated by ROS in cardiomyocytes. Studies have proved that lead (Pb) can affect the balance of mitochondrial dynamics by inducing oxidative stress, resulting in autophagy, which ultimately causes the chicken spleen immune dysfunction (Han et al., 2017). As2O3 could trigger extrinsic and intrinsic apoptosis pathways in immune organs of chickens, meanwhile, ROS generated by oxidative stress might be an important driver of excessive apoptosis (Zhao et al., 2017a). Manganese (Mn) induced oxidative stress and subsequent DNA damage and apoptosis in testicular tissues of chickens (Liu et al., 2013b). Whereas, adverse effects of As2O3 exposure to testicular tissues of chickens have not been explored before. Therefore, it is necessary to research the mechanism of testes exposure to As2O3 and the cross-talk between apoptosis and autophagy in this study. Testes are important reproductive organs of chickens, which are related to the production and reproduction of the poultry industry. If the testicles cause poisoning, the quality of sperm in the testes will be affected, eventually leading to sperm dysplasia or deformity, which will affect the success rate of mating. Or even if the mating is successful, the offspring will be stunted, which will affect the meat quality and harm the development of the breeding industry. In addition, As is now a ubiquitous poisonous metalloid and cases of excessive As have been reported repeatedly. So it makes more sense to study the effects of As on the testicles of chickens. We have previously studied the effects of As2O3 poisoning on testicular inflammation (Sun et al., 2017b), in this experiment, we use the histopathology, Transmission electron microscopy (TEM) and TdT-mediated dUTP nick end labeling (TUNEL) assay to observe the ultrastructural and apoptosis index of the testes, detect the CAT, GSH, MDA and ability of anti-hydroxy radical (AHR) levels to analyze oxidative stress, utilize Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot to assay the expressions of autophagy- and apoptosis-related genes. Reveal the effects of A2O3 on testicular poisoning in chickens, including disordered antioxidant system, increased apoptosis and autophagy, the cross-talk between apoptosis and autophagy, and slightly tissues damage. MATERIALS AND METHOD Animals and Treatment All procedures used in the present study were approved by the Institutional Animal Care and Use Committee of Northeast Forestry University in Harbin, China (approval no. UT-31; 20 June 2014). Seventy-two 1-day-old male Hy-line chickens, (purchased by Weiwei Co. Ltd. Harbin, China) were divided into four groups at random: the control group (C group), the low-As2O3 group (L group), the middle-As2O3 group (M group) and the high-As2O3 group (H group) (18 chickens/group). Throughout the experimental period, feed and water were allowed ad libitum. Feeding, housing and handling of experimental animals were conducted according to the established commercial managemental practices. The basal diet was formulated according to the National Research Council (NRC) (1994). The composition of the basal diet for chickens was 2.766% metabolizable energy (kcal/kg), 18.45% crude protein, 2.82% crude fiber, 3.80% calcium, and 0.62% phosphorus. Treatment groups were maintained on a low-As2O3-sufficient diet (L group) containing 0.625 mg/kg body weight (BW), corresponding 7.5 mg/kg feed, a middle-As2O3-sufficient diet (M group) containing 1.25 mg/kg BW, 15 mg/kg feed, and a high-As2O3-sufficient diet (H group) containing 2.5 mg/kg BW, 30 mg/kg feed. The highest dose of sub-chronic toxicity test can use 1/20 to 1/5 of the median lethal dose [Supplementary Appendix 1], and the median lethal dose of As for chicken was 50 mg/kg BW [Supplementary Appendix 2]. As2O3 was purchased from the New Technology Development Company, CHINA AGR UNIV. Six chickens of each group were selected randomly and euthanized by sodium pentobarbital after stress termination on days 30, 60 and 90 of the experiment, respectively. Then, the testes were quickly excised and rinsed with ice-cold 0.9% NaCl solution and frozen immediately in liquid nitrogen, and stored at −80°C until required (Liu et al., 2013a). Histological Observations The samples of testes were prepared as follows: dehydrating, clearing, embedding, baking. Finally, the sections (5 mm) were stained with hematoxylin and eosin and observed. Transmission Electron Microscopy The testis tissues (1 mm3) were rapidly extracted from the typical sample in all H groups. TEM was performed as described previously (Liu et al., 2016). TUNEL Assay Testis tissues processing for apoptosis-related DNA fragmentation were evaluated by TUNEL Assay using the In situ Cell Death Detection kit (Roche Diagnostics, Shanghai, China) according to the manufacturer's instruction. Results were expressed as the percentage of TUNEL-positive cells among the total number of cells counted (Yao et al., 2013a). Determination of Antioxidant System CAT, GSH, MDA, SOD, GPx, total antioxidant capacity (T-AOC) and ability of AHR levels in testis tissues (n = 5/group) indices indicated in the figures were detected using detection kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's protocol. Determination of the mRNA Expression of Autophagy- and Apoptosis-related Genes by RT-qPCR Total RNA was isolated from testis tissue samples (50 mg tissue; n = 5/group) were isolated from synchronized populations by using Trizol reagent (Takara, Japan). cDNA was created by the PrimeScriptTM RT Reagent Kit (Takara, Japan) and used for qPCR analysis, afterwards. Specific primers used for amplification were shown in Supplementary Appendix 3. The details were indicated in previous research (Li et al., 2017). Table 1. Body weights and testicular weights after As2O3 exposure.   Body weight (g)  Testis weight (g)  Testis weight/Body weight (%)  C group  1600 ± 14.7  4.40 ± 0.10  0.275  L group  1600 ± 16.1  2.03 ± 0.16*  0.127*  M group  1560 ± 20.0  1.51 ± 0.05*  0.097*  H group  1580 ± 15.9  1.14 ± 0.12**  0.072**    Body weight (g)  Testis weight (g)  Testis weight/Body weight (%)  C group  1600 ± 14.7  4.40 ± 0.10  0.275  L group  1600 ± 16.1  2.03 ± 0.16*  0.127*  M group  1560 ± 20.0  1.51 ± 0.05*  0.097*  H group  1580 ± 15.9  1.14 ± 0.12**  0.072**  Values represent the mean S.D. *P < 0.05, **P < 0.01, n = 5. View Large Table 1. Body weights and testicular weights after As2O3 exposure.   Body weight (g)  Testis weight (g)  Testis weight/Body weight (%)  C group  1600 ± 14.7  4.40 ± 0.10  0.275  L group  1600 ± 16.1  2.03 ± 0.16*  0.127*  M group  1560 ± 20.0  1.51 ± 0.05*  0.097*  H group  1580 ± 15.9  1.14 ± 0.12**  0.072**    Body weight (g)  Testis weight (g)  Testis weight/Body weight (%)  C group  1600 ± 14.7  4.40 ± 0.10  0.275  L group  1600 ± 16.1  2.03 ± 0.16*  0.127*  M group  1560 ± 20.0  1.51 ± 0.05*  0.097*  H group  1580 ± 15.9  1.14 ± 0.12**  0.072**  Values represent the mean S.D. *P < 0.05, **P < 0.01, n = 5. View Large Western Blot Analysis of Autophagy- and Apoptosis-related Genes Protein extracts were prepared by using SDS Lysis Buffer, protein concentration was quantified using Enhanced BCA Protein Assay Kit (Beyotime, China) according to the manufacturer's protocol. Proteins were separated by electrophoresis through 15% SDS-polyacrylamide gels and transferred to PVDF membranes (Yao et al., 2013b). The membranes were incubated with diluted primary antibodies against LC3 I/II (1:1000, Wanleibio, China), dynein (1:5000, Abcam, USA), Beclin-1 (1:1000, Novusbio, USA), Autophagy associated gene 5 (ATG5)/ATG4B (1:500, ABclonal, China), TORC1/Cytochrome c (Cyt c) (1:500, Bioss, China), Bcl-2/Caspase-9/Bcl-2 Associated X Protein (Bax)/p53/Caspase-3 (1:1000, Wanleibio, China), Caspase-8/anti -β-actin monoclonal antibody (1:2000, proteintech, China). Subsequently, horseradish peroxidase-conjugated secondary antibody were used to detect specific reaction products. The details were indicated in previous research (Zhao et al., 2017a). Statistical Analysis The experimental data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 5.0 software. SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was utilized for all the statistical analyses conducted in this study. If there were significant differences between treated groups and the control group, a one way ANOVA was used to assess them. Differences between means were assessed using Tukey's paired test between the diverse treated groups. P values less than 0.05 were considered to indicate statistically significant. RESULTS Testis and Body Weights As shown in Table 1, on 90 days post-treatment, body weight was similar between chickens in all groups. The testes of As2O3 treated animals were significantly smaller than those of the control chickens (P < 0.05, P < 0.01), reflecting the generalized toxicity of this compound. The ratios of testis weight to body weight decreased (P < 0.05, P < 0.01) in all treatment groups, compared to the control group. Histological Analysis The results of histological observations of testes on day 90 are shown in Figure 1. Compared to the C group (Figure 1a), part of spermatogenic cells including spermatogonium, primary spermatocyte, secondary spermatocyte and spermatid in the seminiferous tubules were vacuolar and degenerative in L, M and H groups (Figure 1b–d). Figure 1. View largeDownload slide Histopathological changes in the testis tissues of chickens (×400). a. C group. b. L group. c. M group. d. H group on 90 day. Blue, green, yellow and red arrows in C group indicate spermatogonium, primary spermatocyte, secondary spermatocyte and spermatid, respectively. And in L, M and H groups, the same colored arrows represent the corresponding diseased cells, respectively. Figure 1. View largeDownload slide Histopathological changes in the testis tissues of chickens (×400). a. C group. b. L group. c. M group. d. H group on 90 day. Blue, green, yellow and red arrows in C group indicate spermatogonium, primary spermatocyte, secondary spermatocyte and spermatid, respectively. And in L, M and H groups, the same colored arrows represent the corresponding diseased cells, respectively. Ultrastructural Analysis As shown in Figure 2, some representative pictures of testis tissues samples on days 30, 60 and 90 of the experiment in H group illustrated the As-induced ultrastructural changes in testis tissues. Transmission electron microscopy revealed normal testicular ultrastructure in C group (Figure 2a). In stark contrast, As2O3 exposure caused serious abnormalities in testis tissues. Typical characteristics of apoptosis, such as condensation and margination of nuclear chromatin were also found, as well as swollen mitochondria with the degeneration or loss of cristae. In addition, numerous autophagosomes-like structures were obviously presented in the testes of As2O3-treated groups, compared to the C group (Figure 2b–d). Figure 2. View largeDownload slide Developmental exposure to As induced ultrastructural abnormalities in chicken testes. Representative transmission electron microscopy images of testicular tissues from a. C group. b. H group on 30 day, c. 60 day and d. 90 day. Double arrows: typical characteristics of apoptosis; fold arrows: swollen mitochondria; thin arrows: autophagosomes, n = 5. Figure 2. View largeDownload slide Developmental exposure to As induced ultrastructural abnormalities in chicken testes. Representative transmission electron microscopy images of testicular tissues from a. C group. b. H group on 30 day, c. 60 day and d. 90 day. Double arrows: typical characteristics of apoptosis; fold arrows: swollen mitochondria; thin arrows: autophagosomes, n = 5. TUNEL Assay The results of TUNEL assay were showed in Figure 3. Normal cell apoptosis appeared in the testis tissues of the C group (Figure 3a). But the number of apoptosis cells was significantly increased in H groups on days 30, 60 and 90, compared with corresponding C group (P < 0.05, P < 0.01) (Figure 3b–e). Figure 3. View largeDownload slide Representative images of TUNEL stained testicular cross sections from a. C group. b. H group on 30 day, c. 60 day and d. 90 day (×400). e. Apoptosis index. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Figure 3. View largeDownload slide Representative images of TUNEL stained testicular cross sections from a. C group. b. H group on 30 day, c. 60 day and d. 90 day (×400). e. Apoptosis index. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Results of Analysis of Oxidative Stress and Antioxidant Indices CAT, GSH, MDA, SOD, GPx, T-AOC and ability of AHR levels in the chicken testes on days 30, 60 and 90 are presented in Figure 4. In general, the activities of CAT significantly decreased (P < 0.05) in all treatment groups on days 30, 60 and 90, expect the L group on day 30 (P > 0.05) (Figure 4a). The activities of GSH declined remarkably (P < 0.05) in all treatment groups on days 60 and 90, and decreased in H group on day 30, however, had no significant change in L and M group (P > 0.05) (Figure 4b). The GPx levels were decreased in all treatment groups on days 60 and 90 (P < 0.05, P < 0.01), however, it had no significant change on day 30 (Figure 4g). T-AOC, SOD and the ability of AHR were decreased in all treatment groups (P < 0.05, P < 0.01), compared to the corresponding C group (Figure 4c, e, f). Whereas, the concentrations of MDA content were significantly increased in all treatment groups on day 90 and in H group on days 30 and 60 (P < 0.05) (Figure 4d). Figure 4. View largeDownload slide Results of analysis of oxidative stress and antioxidant indices in the testis tissues of chickens. a. Represented the results of CAT. b. Represented the results of GSH. c. Represented the results of AHR. d. Represented the results of MDA contents. e. Represented the results of T-AOC. f. Represented the results of SOD. g. Represented the results of GPx. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Figure 4. View largeDownload slide Results of analysis of oxidative stress and antioxidant indices in the testis tissues of chickens. a. Represented the results of CAT. b. Represented the results of GSH. c. Represented the results of AHR. d. Represented the results of MDA contents. e. Represented the results of T-AOC. f. Represented the results of SOD. g. Represented the results of GPx. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Effects of As2O3 on Apoptosis-related Genes’ mRNA and Protein Levels in the Testis Tissues of Chickens Effects of As2O3 treatment on the levels of the apoptosis-related genes’ mRNA and proteins (p53, Bcl-2, Bax, Cyt c, Caspase-9, Caspase-3, Caspase-8) in the chicken testes were showed in Figure 5. Compared with the C group, the treatment of As2O3 in H group was significantly upregulated the expressions of all the detected apoptosis-related genes’ mRNA and protein (P < 0.05, P < 0.01), except the expression of Bcl-2. The mRNA and protein levels of Bcl-2 were downregulated markedly, compared to the corresponding C group (Figure 5a–c) (P < 0.05). Figure 5. View largeDownload slide Effects of apoptosis-related genes’ mRNA and protein levels in the testis tissues of chickens. a. Represented the mRNA levels of apoptosis-related genes in the testes. b and c Represented the protein levels of apoptosis-related genes. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Figure 5. View largeDownload slide Effects of apoptosis-related genes’ mRNA and protein levels in the testis tissues of chickens. a. Represented the mRNA levels of apoptosis-related genes in the testes. b and c Represented the protein levels of apoptosis-related genes. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Effects of As2O3 on Autophagy-related Genes’ mRNA and Protein Levels in the Testis Tissues of Chickens Effects of As2O3 treatment on the levels of the autophagy-related genes’ mRNA and proteins in the chicken testes were showed in Figure 6. Compared with the C group, the mRNA and protein levels of ATG5, ATG4B, Beclin-1, dynein and LC3 II/I presented higher levels in H group (P < 0.05, P < 0.01), in a dose-dependent manner. However, the mRNA and protein levels of TORC1 were markedly decreased, compared to the corresponding C group (Figure 6a–c) (P < 0.05, P < 0.01). Figure 6. View largeDownload slide Effects of autophagy-related genes’ mRNA and protein levels in the testis tissues of chickens. a. Represented the mRNA levels of autophagy-related genes in the testes. b and c. Represented the protein levels of autophagy-related genes. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. Figure 6. View largeDownload slide Effects of autophagy-related genes’ mRNA and protein levels in the testis tissues of chickens. a. Represented the mRNA levels of autophagy-related genes in the testes. b and c. Represented the protein levels of autophagy-related genes. Error bars represent S.D. *P < 0.05, **P < 0.01, n = 5. DISCUSSION Arsenic is considered as the most harmful substance in the Agency for Toxic Substances and Disease Registry (Zhao et al., 2017b). Numerous reports have shown that chronic As2O3 exposure can cause the damages of the organisms, i.e., skins, lungs and bladder cancers, cardiovascular dysfunction, adverse pregnancy outcomes, cognitive deficits and type-2 diabetes (Abdul et al., 2015). Previous researches have suggested that As2O3 induced testicular toxicity that can cause inflammatory and heat shock responses in chickens (Sun et al., 2017b). In this research, we aim to analyze the correlation between the exposure to As2O3 and testicular ultrastructural changes, oxidative stress, apoptosis and autophagy, as well as the cross-talk between apoptosis and autophagy after As2O3 exposure. Testis weight, the ratios of testis weight, testicular histopathology and TUNEL assays (Table 1, Figures 2 and 3) were utilized to appraise the extent of cell death resulting from As2O3 treatment; showing vacuolar and degenerative spermatogenic cells and apoptotic cells in the treatment groups which meant the degeneration and necrosis of cells and the decline of the testicular function. Ultrastructural analysis and TUNEL assay have intuitively demonstrated that As2O3 exposure to testes can induce apoptosis and autophagy (Figures 2 and 3) in a time-dependent manner. Since oxidative stress can induce apoptosis and autophagy, we have conducted a correlation analysis of oxidative damage. Antioxidant enzyme activities in the testes of chickens varied with As2O3 concentrations and duration of As2O3 exposure. Likewise, a significantly dose-dependent decrease of GSH was found during the exposure. In chickens, the activities of antioxidant enzymes, such as GPx and CAT, were inhibited after As2O3 exposure depending on exposure time and concentration in immune organs and brain tissues (Zhao et al., 2017a; Zhao et al., 2017b). CAT has the effect of catalyzing the decomposition of highly toxic hydrogen peroxide (H2O2) into water and molecular oxygen (Adeyemi et al., 2015). SOD removes superoxide radicals by converting them into H2O2, which is rapidly converted into water by CAT. SOD and CAT work together to eliminate ROS, and the SOD-CAT system forms the first line of defence against oxidative stress (Wafa et al., 2011). The major function of GPx is reducing soluble H2O2 and converting it into water in the presence of oxidated glutathione (Sun et al., 2016). In the present study, the CAT, SOD and GPx activities were reduced after As2O3 exposure, which means the reduced detoxification capacity. GSH is known to play a role in protecting cells from ROS damage through metal toxicity (Hellou et al., 2012). In this study, the level of GSH is depleted after As2O3 exposure, which increased the susceptibility of the testes to free radical-induced toxicity. Simultaneously, the ability of AHR is down-regulated in a dose-dependent fashion. What's more, the level of T-AOC decreased, which was more indicative of the decline in the testicular antioxidant capacity. In addition, MDA is a product of lipid peroxidation, which is widely used to assess oxidative damage in various environmental attacks (Wang et al., 2008). MDA content increased in chickens brain tissues exposure to As2O3 (Zhao et al., 2017b). According to previous studies, with the increase of dietary As content, the content of MDA gradually increased in this study, indicating that the lipid peroxidation and greater extent of oxidative damage were enhanced in As2O3-exposed models. Previous reports indicate that oxidative stress induced by NaAsO2 may lead to apoptosis and necrosis in rat proximal tubular cells (Prabu and Muthumani, 2012). As2O3 can induce oxidative stress, and cause autophagy, finally lead to the neurotoxicity in chicken brain tissues (Sun et al., 2017a). However, in chickens, it is not well clear whether the As2O3-induced testes undergo apoptosis and/or autophagy. Accordingly the current study was designed to understand the mechanistic approach adopted by testes in response to As2O3-induced in chicken, and to observe the cross-talk between apoptosis and autophagy. Apoptosis is a programmed cell death in which the specific signaling pathways (extrinsic and intrinsic apoptosis pathways) are activated. It is well known that important apoptosis regulatory mechanism includes death receptors, caspases, mitochondria, Bcl-2 and tumor-suppressor genes are well known as important apoptosis regulatory mechanisms (Alamolhodaei et al., 2015). Apoptosis induced by NaAsO2 depends on exposure time and concentration (Chen et al., 1998). In this study, As2O3-induced apoptosis was mediated through the increases of caspases activation, p53 and the corresponding changes of Bcl-2 gene family. It is reported that oxidative stress may trigger apoptosis (Xie et al., 2017). Oxidative stress accumulates too much •OH that cannot be cleared by the body later they cause DNA damage. What's more, DNA damage can increase the level of p53 which can regulate gene expression of Bcl-2 gene family (Yu et al., 2001). A pro-apoptotic member of the Bcl-2 gene family called Bax increased significantly in a dose-dependent fashion. Meanwhile, Bcl-2, the anti-apoptotic member was declined after As2O3 exposure in the current study. Increased Bax further stimulated Cyt c, thereby activated Caspase-9 up-regulation, and finally generated Caspase-3, which led to apoptosis (Eisenberg-Lerner et al., 2009). This is a pathway to cause apoptosis called the intrinsic pathway. In this study, after As2O3 exposure, intrinsic pathway of apoptosis-related genes’ mRNA and protein levels such as Cyt c, p53, Caspase-9 Caspase-3 and Bax were increased as a dose dependent fashion, compared to the corresponding C group, which meant intrinsic pathway of apoptosis was activated by As2O3 exposure. In addition, the extrinsic pathway of apoptosis was also activated. In the present study, Fas is activated after oxidative stress, which can be associated with caspase activation, such as Caspase-8 and Caspase-3 (Wajant, 2002). Caspase-3 is a key effector molecule for apoptosis execution. Most factors that trigger apoptosis eventually require Caspase-3-mediated signal transduction pathways, leading to apoptosis (Rong et al., 2008). Our study demonstrated that As2O3 significantly increased Fas, Caspase-8 and Caspase-3 expressions in a dose-dependent fashion, which means extrinsic pathway of apoptosis is activated. What's more, we found that numerous factors (Cyt c, p53, Bax, Bcl-2, Caspase-9) controlled intrinsic apoptosis, which should be more notable in this study. Autophagy is a cellular degradation pathway which used to remove damaged or redundant proteins and organelles. It is often thought of as a natural process for maintaining cellular homeostasis and the reaction of cells to the progress of starvation, infection or disease (Chen et al., 2015). The study has pointed out that atrazine and chlorpyrifos and their combination can induce oxidative stress and autophagy in the immune organs of common carp (Chen et al., 2015). Accumulating evidence indicates that Beclin-1 participates in the early stages of autophagosome formation, facilitating the nucleation of autophagosomes (Kihara et al., 2001). Our research revealed that the mRNA and the protein levels of Beclin-1 were increased. Cao et al. (2009) showed that the mRNA of Beclin-1 is increased significantly in rats after pilocarpine-induced status epilepticus. Our results show that Beclin-1 has similar changes in the level of stress response in birds as in mammals. LC3 and dynein are widely used to monitor autophagy. Under the stimulus of autophagy, LC3 converts its soluble cytoplasmic form (LC3-I) into the form of autophagy associated with membrane expansion (LC3-II). The ratio of LC3-II/LC3-I was used to evaluate the number of autophagosomes (Qu et al., 2015). Our results showed that after As2O3 exposure, the mRNA and protein levels of the ratio of LC3-II/LC3-I up-regulated, meanwhile, the mRNA and protein levels of dynein were increased significantly, compared to the corresponding C group, supporting the presence of enhanced autophagy in chickens testes exposed to As2O3. Besides, ATG4B and ATG5 are also involved in the formation of autophagosomes, which contact Beclin-1 with LC3 and dynein (Eisenberg-Lerner et al., 2009). This research showed that the levels of ATG4B and ATG5 were both up-regulated in all As2O3-treated groups, compared to the corresponding C group. Eisenberg-Lerner et al. (2009) manifested that apoptosis and autophagy are not only mutually antagonistic pathways, but also show a synergistic effect and also offset each other. Therefore, we explore the cross-talk between apoptosis and autophagy in this study. mTOR plays a crucial positive moderating role in cell growth, proliferation, cell cycle and so on. The function of mTOR is primarily mediated by mTORC1 and mTORC2. The occurrence of autophagy is mainly regulated by mTORC1, therefore, we explore the changes in TORC1 after As2O3 exposure (Wei et al., 2012). Autophagy activity was negatively regulated by mTOR. In current study, the levels of TORC1 declined remarkably in all As2O3-treated groups, compared to the corresponding C group, which meant autophagy was activated. What's more, there are studies show that mTOR can stimulate the expression of Beclin-1, simultaneously, Beclin-1 is a main regulator of autophagy, and several proteins such as Bcl-2 family members interact with it (Qu et al., 2015). That's already mentioned that Beclin-1 can bind Bcl-2/Bcl-XL to form constitutive Bcl-2/Bcl-XL-Beclin-1 to reduce Beclin-1 monomeric to inhibit autophagy. However, in current study, the levels of Beclin-1 were highly increased, and Bcl-2 levels were inhibited in a dose-dependent fashion, which meant Beclin-1 monomeric increased and apoptosis and autophagy were both activated. Besides, the inhibition of mTOR is also regulated by p53. In current study, the levels of p53 were markedly increased in a dose-dependent fashion, which also suppressed the level of mTOR and promoted autophagy. To sum up, Bcl-2 genes family and p53, which are apoptosis-related genes, can adjust autophagy by regulating the level of mTOR, which in turn acts on apoptosis-related genes to regulate apoptosis. In a word, apoptosis and autophagy can promote each other in testis tissues of chickens after As2O3 exposure. CONCLUSION To sum up, the findings in the present study provide some preliminary evidences that exposure to As2O3 can cause tissue damage in testes of chickens, which will affect the breeding of the poultry industry and people's economic benefits. 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Poultry ScienceOxford University Press

Published: May 14, 2018

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