Biometals (2015) 28:255–265 DOI 10.1007/s10534-014-9819-3
Roles of oxidative stress and endoplasmic reticulum stress in selenium deficiency-induced apoptosis in chicken liver Linlin Yao • Qiang Du • Haidong Yao Xi Chen • Ziwei Zhang • Shiwen Xu
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Received: 16 September 2014 / Accepted: 26 December 2014 / Published online: 14 March 2015 Ó Springer Science+Business Media New York 2015
Abstract Oxidative stress and endoplasmic reticulum (ER) stress are involved in different types of stressinduced injuries. The aim of the present study was to evaluate the effect of Se deficiency on oxidative stress, ER stress and apoptosis in chicken livers. Chickens (1 day old, n = 180) were randomly divided into two groups: the L group [fed with a Se-deficient (Se 0.033 mg/kg) diet] and the control group [fed with a normal (Se 0.2 mg/kg) diet]. Factor-associated oxidative stress, catalase (CAT) activity, H2O2 production and the inhibition of hydroxyl radicals (OH) in the chicken liver were determined on days 15, 25, 35, 45, 55 and 65, respectively. In addition, ER stress-related genes (GRP78, GRP94, ATF4, ATF6 and IRE) and apoptosis-related genes (caspase3 and Bcl-2) were examined by fluorescence quantitative PCR or western blot analysis. Apoptosis levels were also measured using ultrastructural observations and the TdT-mediated dUTP nick end labeling assay. The results showed that CAT activity and OH inhibition were decreased and that H2O2 production was increased in the low-Se group, which demonstrated that oxidative stress occurred in the
L. Yao Q. Du H. Yao X. Chen Z. Zhang (&) S. Xu (&) Department of Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, People’s Republic of China e-mail: [email protected] S. Xu e-mail: [email protected]; [email protected]
chicken liver. The ER stress-related genes (GRP78, GRP94, ATF4, ATF6 and IRE) and the apoptosisrelated gene caspase3 were increased (p \ 0.05), while Bcl-2 was decreased (p \ 0.05) by Se deficiency. In addition, apoptosis and ER lesions were observed by ultrastructural observations of the chicken liver in the low-Se group. The level of apoptosis and the number of apoptotic cells increased with time. These results indicated that the oxidative-ER stress pathway participates in Se deficiency-induced apoptosis in the chicken liver. Keywords Selenium deficiency Oxidative stress Endoplasmic reticulum stress Apoptosis Chicken liver
Introduction Selenium (Se) deficiency is associated with numerous diseases. Se implements its biological function through incorporation into selenoproteins. In addition, Se deficiency caused injuries in several different chicken organs, including the brain (Sheng et al. 2014; Xu et al. 2013), muscles (Yao et al. 2013a, b), the gastrointestinal tract (Gao et al. 2012), the spleen (Yu et al. 2011) and the liver (Sun et al. 2011). The study of Fischer showed that the liver contains a high concentration of Se, which participates in many biological processes that range from cellular antioxidant defense to the protection and repair of DNA to apoptosis
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(Fischer et al. 2006). Se deficiency could induce liver necrosis in rats (Matsuda et al. 1998), hepatosis dietetica in pigs (Doyle et al. 2011), oxidative damage in the livers of patients with HCV-related CLD (Ko et al. 2005) and apoptosis in rat livers (Miao et al. 2013). Oxidative stress is described as an imbalance between reactive oxygen species (ROS) generation and antioxidant capacity, which triggers apoptosis through a variety of signaling pathways, such as the ER stress response (Kitamura and Hiramatsu 2010). ER stress is considered an early or initial response of cells to stress or damage (Doyle et al. 2011). The unfolded protein response (UPR) occurs in response to disturbed ER homeostasis and is induced to restore the protein folding capacity of ER through the accumulation of unfolded proteins in the ER lumen, which triggers both cell protective and cell death responses (Srivastava et al. 2013). GRP78 is a central regulator for ER stress due to its role as a major ER chaperone with anti-apoptotic properties as well as its ability to control the activation of transmembrane ER stress sensors (IRE1, PERK, and ATF6) through a binding-release mechanism (Hammadi et al. 2013; Lee 2005). ER stress is also related to the occurrence of apoptosis, in which CHOP, TRB3 (Zhang et al. 2014) and other apoptosis related genes (proapoptotic (caspase) and anti-apoptotic (Bcl-2 related family) are involved (Jergens et al. 2014). Previous studies showed that ER stress plays a key role in the regulation of apoptosis caused by a variety of toxic insults, including ROS chemicals and heavy metals. In chickens, Se deficiency causes oxidative damage in the brain (Xu et al. 2013) and immune tissues (Zhang et al. 2013). The study of Irmak showed that selenium deficiency-induced apoptosis in HCCderived cell lines was caused by oxidative stress (Irmak et al. 2003). In addition, Se deficiency could induce apoptosis in MDBK cells (Kayanoki et al. 1996) and NIH3T3 cells (Zhou et al. 2003). Therefore, diseases or damage related to Se deficiency may be mediated by oxidative-ER stress or apoptosis. However, the exact mechanism is still unclear. It has not been reported whether Se deficiency induced oxidative stress or ER stress or how these pathways regulate apoptosis in the chicken liver. In the present study, we examined factor-associated oxidative stress (CAT activity, H2O2 production and OH inhibition), ER stress (GRP78, GRP94, ATF4, ATF6 and IRE), and apoptosis (the apoptosis-associated markers caspase3
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and Bcl-2, ultrastructural observations and TUNEL assay) in chicken livers.
Materials and methods Animal care and experimental design All procedures used in the present study were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. Chickens (n = 180, 1-day-old, laying hens, Weiwei Co. Ltd., Harbin, China) were randomly divided into two groups (90 chickens per group). The chickens were maintained on either a Se-deficient granulated diet (L group, Se-deficient granulated diet including corn, soybean meal and wheat bran from Longjiang County, the Se deficiency region of Heilongjiang Province in China, and a blend of materials that did not add Se (Weiwei Co. Ltd., Harbin, China)) or the commercial granulated diet (Control group, Weiwei Co. Ltd., Harbin, China) for 15, 25, 35, 45, 55, or 65 days. The Se content of the Se-deficient granulated diet and the basal commercial granulated diet were analyzed by GB/T 13,883-2008 (PONY TEST Co., Beijing, China), which contained 0.033 and 0.200 mg/ kg Se (sodium selenite), respectively. The feed and water were supplied and libitum. Following euthanasia with sodium pentobarbital, livers (15, 25, 35, 45, 55 and 65 days old) were quickly collected and frozen immediately in liquid nitrogen and stored at -80 °C until required. Ultrastructural observations For electron microscopy, liver tissue specimens were fixed with 2.5 % glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 3 h at 4 °C, washed in the same buffer for 1 h at 4 °C and post fixed with 1 % osmium tetroxide in sodium phosphate buffer for 1 h at 4 °C. The tissues were then dehydrated in graded series of ethanol, 50, 70, 90 and 100 %, for 10 min each, and incubated twice in propylene oxide (100 % ethanol: 100 % propylene oxide = 1: 1 and 100 % propylene oxide for 10 min each at 4 °C). The tissue specimens were embedded in araldite. Ultrathin sections were stained with Mg-uranyl acetate and lead citrate for transmission electron microscope evaluation.
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Measurement of apoptosis by TUNEL assay TUNEL was carried out to analyze DNA fragmentation indicative of cellular apoptosis using the in situ cell death detection kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s protocol. Paraffin wax-embedded tissue sections were treated with proteinase K and the endogenous peroxidase activity was blocked with hydrogen peroxide. The sections were incubated at 37 °C with the terminal TdT nucleotide mixture for 1 h. Then, the reaction was stopped and the slides were rinsed with phosphate buffered saline (PBS). Nuclear labeling was developed with horseradish peroxidase and diaminobenzidine. Hematoxylin was used for counterstaining. The number of apoptotic cells in each slide from at least five different fields were analyzed using the Image-Pro Plus software (version 6.0 for Windows) with the aid of a microscope (BA400, Motic, China). The number of positive cells was averaged for statistical analysis. CAT activity, H2O2 production and OH inhibition assays Assays for CAT activity, H2O2 production and OH inhibition were performed at each time point using liver homogenates and specific kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions. Western blot analysis Protein extracts were subjected to 15 % SDS-polyacrylamide gel electrophoresis under reducing conditions. The separated proteins were then transferred to nitrocellulose membranes for 2 h at 100 mA in a tank transfer apparatus containing Tris-glycine buffer and 20 % methanol. Membranes were blocked with 5 % skim milk for 24 h and incubated overnight with diluted primary antibodies against Bcl-2 (1:500, Santa Cruz Biotechnology, USA), caspase3 (1:100, Santa Cruz Biotechnology, USA) and GRP78 (1:1,000, polyclonal antibody produced by our lab) followed by a horseradish peroxidase (HRP)-conjugated secondary antibody against goat (Bcl-2) or rabbit (caspase3 and GRP78) IgG (1:1,000, Santa Cruz Biotechnology, USA). To verify equal loading of the samples, the membrane was incubated with a
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monoclonal b-actin antibody (1:1,000, Santa Cruz Biotechnology, USA), followed by a HRP-conjugated goat anti-mouse IgG (1:1,000) secondary antibody. The signal was detected with X-ray films (TransGen Biotech Co., Beijing, China). The optical density (OD) of each band was determined using an Image VCD gel imaging system, and the relative abundance of the Bcl2, caspase3 and GRP78 proteins were expressed as the ratios of OD of each of these proteins to that of b-actin. Quantitative real-time PCR analysis Total RNA was isolated from tissue samples using Trizol reagent, according to the manufacturer’s instructions (Invitrogen, Shanghai, China). The concentration and purity of the total RNA were determined spectrophotometrically at 260 and 280 nm using a GeneQuant 1300. The procedure of the reverse transcription was performed according to the manufacturer’s instructions (Invitrogen, Shanghai, China). Synthesized cDNA were stored at -20 °C for PCR. Oligo 7.0 Software was used to design specific primers for GRP78, GRP94, Bcl-2, caspase3, ATF4, ATF6, IRE and GADPH (a housekeeping gene used as an internal reference) based on known chicken sequences (Table 1). Primers were synthesized by Invitrogen Biotechnology Co. Ltd. in Shanghai, China. Quantitative real-time PCR was performed on an ABI PRISM 7500 Detection System (Applied Biosystems, Foster City, CA). Reactions were performed in a 20 lL reaction mixture containing 10 lL of the 29 SYBR Green I PCR Master Mix (TaKaRa, China), 2 lL cDNA, 0.4 lL of each primer (10 lM), 0.4 lL of the 509 ROX reference Dye II and 6.8 lL of PCRgrade water. The PCR procedure for GRP78, GRP94, Bcl-2, caspase3, ATF4, ATF6, IRE and GADPH consisted of 95 °C for 30 s followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 60 °C for 30 s. Dissociation curves were analyzed by Dissociation Curve 1.0 Software (Applied Biosystems) for each PCR reaction to detect and eliminate possible primerdimers and non-specific amplification. The relative abundance of each mRNA was calculated according to the method of Pfaffl (2001). Statistical analysis on all data was performed using GraphPad Prism 5.0 software, and all data were assessed using one-way ANOVA. Differences between the means were assessed using Tukey’s honestly significant
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Table 1 Gene-specific primers used in the real-time quantitative reverse transcription PCR Gene
Primer sequence
GRP 78
Forward 50 -GAATCGGCTAACACCAGAGGA-30 Reverse 50 -CGCATAGCTCTCCAGCTCATT-30
GRP94
Forward 50 -CAAAGACATGCTGAGGCGAGT-30 Reverse 50 -TCCACCTTTGCATCCAGGTCA-30
Bcl-2
Forward 50 -ATCGTCGCCTTCTTCGAGTT-30 Reverse 50 -ATCCCATCCTCCGTTGTCCT-30
Caspase3
Forward 50 -CATCT GCATCC GTGCCTGA-30 Reverse 50 -CTCTCGG CTGTGGTGGTGAA-30
ATF4
Forward 50 -GAATCGGCTAACACCAGAGGA-30 Reverse 50 -CGCATAGCTCTCCAGCTCATT-30
ATF6
Forward 50 - CGTCGTCTGAACCACTTACTGA-30 Reverse 50 -CCTTCTTTCCTAACAGCCACAC-30
IRE
Forward 50 -CTACAGGTCGCTCCTCACATC-30 Reverse 50 -ATCAGTCCTTCTGCTCCCATCT-30
GADPH
had brown stained nuclei. Livers from the L group had an increased number of apoptotic cells compared to the control group, which further demonstrated that Se deficiency promoted apoptosis in chicken livers (Fig. 2).The effects of Se deficiency on the percent of apoptotic cells in liver were shown in Fig. 3. The percent of apoptotic cells in liver was increased (p \ 0.05) in the L group compared to the control group from 15 to 65 days of age. A significant increase in the percent of apoptotic cells in the liver was observed over time for chickens that were fed a Sedeficient basal diet.
0
0
Forward 5 -AGAACATCATCCCAGCGT-3 Reverse 50 -AGCCTTCACTACCCTCTTG-30
difference test for post hoc multiple comparisons. All data were expressed as the mean ± SD, where p \ 0.05 was considered a statistically significant difference.
Changes in CAT activity, H2O2 production and OH inhibition The effects of Se deficiency on CAT activity and OH inhibition in liver were shown in Fig. 4a, c respectively. These parameters were decreased (p \ 0.05) in the L groups as compared to the control group from 15 to 65 days of age. H2O2 production increased (p \ 0.05) from 25 to 65 days of age (Fig. 4b). Effects of Se deficiency on the expression of ER stress-related genes (GRP78, GRP94, ATF4, ATF6 and IRE) in chicken livers
Results Ultrastructural changes No ultrastructural changes were observed in the liver of control group (Fig. 1a). The mitochondria from the liver of the L group were vacuolated and the cristae (black arrows) had degenerated. The apoptotic cells showed typical condensed nuclei with horseshoe-like or crescent-shaped cytoplasmic organelles of an inconspicuous structure (red arrows) accompanied by nucleus shrinkage (blue arrows). The nuclei and organelles of some cells were unclear (Fig. 1b). The mitochondria from the liver of the L group were surrounded by fractured ER (black arrows) (Fig. 1c). Their rough ER was extended (black arrows) (Fig. 1d). Measurement of cellular apoptosis using the TUNEL assay We performed the TUNEL assay in chicken livers to confirm the occurrence of apoptosis. Apoptotic cells
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The effects of Se deficiency on the levels of GRP78, GRP94, ATF4, ATF6 and IRE mRNA were shown in Fig. 7a–e, respectively. They were increased (p \ 0.05) in the L groups compared to the control group over time. Notably, the levels of GRP78 and GRP94 mRNA in the L group increased by 21.48 and 22.36 fold, respectively, compared to the control group at 65 days of age. Moreover, the relative levels of GRP78 protein were shown in Figs. 5 and 6a and were also increased (p \ 0.05) in the L group compared to the control groups from 15 to 65 days of age. Effects of Se deficiency on the expression of the apoptosis-related genes (Bcl-2 and caspase3) in chicken livers The effects of Se deficiency on the levels of Bcl-2 and caspase3 mRNA were shown in Fig. 7f, g, while the levels of each protein were shown in Figs. 5 and 6b, c, respectively. Bcl-2 was decreased (p \ 0.05) and
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Fig. 1 Effects of Se deficiency on the ultrastructural changes in liver of chickens from the a control group and b–d L group
caspase3 was increased (p \ 0.05) by Se deficiency at both the mRNA and protein levels.
Discussion Se deficiency induced liver injury, apoptosis and necrosis in different types of animals; however, few reports exist for chicken livers, and the exact mechanism of Se deficiency on liver apoptosis remains unclear. In the present study, evident apoptotic characteristics and an increased rate of apoptosis were observed in the liver of Se-deficient chicken compared with the control group over time. The experimental groups were accompanied by oxidative stress and ER stress, which launched the oxidative-ER apoptosis pathway.
Se deficiency has been shown to induce the oxidative stress in different tissues. We previously showed that Se deficiency could cause oxidative damage in chicken brain (Xu et al. 2013) and immune tissues (Zhang et al. 2013). CAT activity, H2O2 production and OH inhibition are the main biological markers of oxidative stress in tissues and organs. CAT is a heme-containing redox enzyme and serves to protect the body from the toxic damage of H2O2 by catalyzing its decomposition into molecular oxygen and water without the production of free radicals, such as OH. According to reports,CAT is closely associated with an increased risk of oxidative stress; ciprofloxacin and enrofloxacin significantly decreased CAT activity compared to the control group (Qin and Liu 2013). Li proved that OH induces apoptosis and oxidative damage in carp erythrocytes (Li et al. 2013).
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Fig. 2 Effects of Se deficiency on the TUNEL assay in chicken livers. a Central veins of the liver in the control group. b Central veins of the liver in the L group. c Portal area of the liver in the control group. d Portal area of the liver in the L group
Fig. 3 Effects of Se deficiency on the apoptotic rate chicken livers. The asterisk indicated that there were significant differences (p \ 0.05) between the control group and the L group at the same time point. Each value represented the mean ± SD of five individuals
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The study of Sun found that the OH was generated in Carassius auratus liver that was exposed to pyrene. The OH production showed a significant increase compared with the control groups (Sun et al. 2008). Liu pointed that the inhibition of OH in the testicles was dose- and time-dependent. There was a daily decrease in the inhibition of OH activity for all experimental groups compared with that of the control group. These results clearly showed that oxidative stress and damage occurred in response to Mn (Liu et al. 2013). In the present study, the activity of CAT was decreased, H2O2 production was increased, and the OH inhibition was decreased in the Se-deficient group compared to the control group. Therefore, similar to prior studies, the present study showed that Se deficiency induced oxidative stress in chicken liver. However, the occurrence of oxidative stress induced continuous ER stress through interfering with
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Fig. 4 Effects of Se deficiency on the CAT activity, H2O2 production and OH inhibition chicken livers. a Effects of Se deficiency on CAT activity. b Effects of Se deficiency on H2O2 production. c Effects of Se deficiency on inhibiting OH ability.
Fig. 5 Effects of Se deficiency on the levels of the GRP78, Bcl2 and caspase3 proteins. The notations 15C, 25C, 35C, 45C, 55C and 65C indicated the control groups from 15 days of age, 25 days of age, 35 days of age, 45 days of age, 55 days of age and 65 days of age, respectively. The notations 15L, 25L, 35L, 45L, 55L and 65L respectively indicated the L groups from 15 days of age, 25 days of age, 35 days of age, 45 days of age, 55 days of age and 65 days of age, respectively
the oxidation of the internal environment of the ER. The latest research also showed for some of the factors involved in the induction of apoptosis, such as tumor necrosis factor (Burton et al. 2009), cigarettes (Tagawa et al. 2008) and cadmium intoxication (Yokouchi et al. 2008), the ER stress response may be downstream of oxidative stress.
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The asterisk indicated that there were significant differences (p \ 0.05) between the control group and the L group at the same time point. Each value represented the mean ± SD of five individuals
When ER stress occurred, chaperone proteins, such as GRP78 and GRP94, are recruited to support the folding of new proteins (Hotamisligil 2010). Therefore, the UPR can enhance the capabilities of the ER to process paraproteins and up-regulate the expression of ER chaperones GRP78 and GRP94 (Lee 2001). A previous study showed that exposure to a pyrrolidine dithiocarbamate (PDTC)/Cu complex could induce cytotoxicity and apoptosis in alveolar epithelial cells via the mitochondria- and ER-stressrelated signaling pathways; during these processes, the expression of ER stress-associated signaling molecules including GRP78, GRP94 and ATF4 were increased by the PDTC/Cu complex (Chen et al. 2010). In our study, the expression GRP78 and GRP94 were significantly elevated in the liver of the L group over time. The up-regulated expression of these proteins clearly indicated that Se deficiency could lead to ER stress. In addition, ER lesions were observed by ultrastructural observations in chicken liver in the low-Se group.
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Fig. 6 Effects of Se deficiency on the levels of the GRP78, Bcl2 and caspase3 proteins in chicken livers. a Effects of Se deficiency on the relative level of GRP78 protein in liver. b Effects of Se deficiency on the relative level of Bcl-2 protein in liver. c Effects of Se deficiency on the relative level of caspase3
protein in liver. The results were from at least three independent experiments. The asterisk indicated that there were significant differences (p \ 0.05) between the control group and the L group at the same time point. Each value represented the mean ± SD
Moreover, upon ER stress, P19 embryonal carcinoma (EC) cells activated various caspases, including caspase3, and exhibited extensive DNA fragmentation (Jimbo et al. 2003). Caspases are key regulators of cell apoptosis,and are involved in ER stressed-induced apoptosis (Fan et al. 2005; Riedl and Shi 2004). Caspase3 activity was significantly increased in treated PC12 cells compared to controls during high glucose-induced apoptosis (Sharifi et al. 2009). On the other hand, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to a decrease in mRNA levels (Liang et al. 2011). The study of Chen also found that Bcl-2 mRNA and protein expressions were decreased in lung epithelial cells during the process of apoptosis induced by ER stress (Chen et al. 2010). Studies have also found that Se deficiency causes cell cycle arrest and increased apoptotic activity (Kim et al. 2004). In present study, both caspase3 mRNA and protein increased and Bcl-2 mRNA and protein levels decreased in the experimental group. In addition, apoptosis was observed by ultrastructural observations and TUNEL assay in chicken liver. These results showed that ER stress is related to the occurrence of apoptosis.
It has been shown that ER stress occurred by several pathways including IRE1, PERK, and ATF6 (Ron and Walter 2007; Sano and Reed 2013; Xu et al. 2005). Among them, the PERK pathway enhances translation of mRNAs, including ATF4 (Armstrong et al. 2010). In a previous study, anacardic acid (6-pentadecylsalicylic acid, AA), which induced apoptosis in pituitary adenoma and lung adenocarcinoma cells through ER stress, not only significantly increased the expression of GRP78, but also increased the expression of ATF4 in both HepG2 and U266 cells (Seong et al. 2013, 2014). IRE1 is derived from the activation of IRE. Considerable evidence has demonstrated a connection between apoptosis and the ER stress and IRE1 pathways (Jiang et al. 2012; Kim et al. 2008). The IRE1 pathway, the most evolutionarily conserved pathway of the three UPR pathways, has a proapoptotic function that involves the activation of GRP78 and IRE1 (Schroder and Kaufman 2005). ATF6 is an ER stress-regulated transmembrane transcription factor that activates the transcription of ER molecular chaperones. In our results, the levels of ATF4, ATF6 and IRE mRNA were increased in the L group compared to the control group. In a word, these
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Fig. 7 Effects of Se deficiency on the levels of GRP78, GRP94, ATF4, ATF6, IRE, Bcl-2 and caspase3 mRNA in chicken livers. a Effects of Se deficiency on the level of GRP78 mRNA in liver. b Effects of Se deficiency on the level of GRP94 mRNA in liver. c Effects of Se deficiency on the level of ATF4 mRNA in liver. d Effects of Se deficiency on the level of ATF6 mRNA in liver. e Effects of Se deficiency on the level of IRE mRNA in liver. f Effects of Se deficiency on the level of Bcl-2 mRNA in liver. g Effects of Se deficiency on the level of caspase3 mRNA in liver. The asterisk indicated that there were significant differences (p \ 0.05) between the control group and the L group at the same time point. Each value represented the mean ± SD of five individuals
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results illustrated that Se deficiency-induced ER stress may be involved in the PERK, ATF6 and IRE1 pathways.
Conclusions The present study showed that Se deficiency led to the occurrence of oxidative stress, ER stress and apoptosis in chicken livers. Moreover, Se deficiency-induced ER stress may be involved in the PERK, ATF6 and IRE1 pathways. Therefore, the oxidative-ER stress pathway participates in the process of Se deficiency-induced apoptosis in the chicken liver. Acknowledgments This study was supported by the National Natural Science Foundation of China (31272626); the International (Regional) Cooperation and Exchange Projects of the National Natural Science Foundation of China (31320103920) and the Study Abroad Foundation of Heilongjiang Province (LC201031). Conflict of interest conflicts of interest.
The authors declare that there are no
References Armstrong JL, Flockhart R, Veal GJ, Lovat PE, Redfern CP (2010) Regulation of endoplasmic reticulum stress-induced cell death by ATF4 in neuroectodermal tumor cells. J Biol Chem 285:6091–6100. doi:10.1074/jbc.M109. 014092 Burton GJ, Yung HW, Cindrova-Davies T, Charnock-Jones DS (2009) Placental endoplasmic reticulum stress and oxidative stress in the pathophysiology of unexplained intrauterine growth restriction and early onset preeclampsia. Placenta 30(Suppl A):S43–S48. doi:10.1016/j.placenta. 2008.11.003 Chen YW et al (2010) Pyrrolidine dithiocarbamate (PDTC)/Cu complex induces lung epithelial cell apoptosis through mitochondria and ER-stress pathways. Toxicol Lett 199:333–340. doi:10.1016/j.toxlet.2010.09.016 Doyle KM, Kennedy D, Gorman AM, Gupta S, Healy SJ, Samali A (2011) Unfolded proteins and endoplasmic reticulum stress in neurodegenerative disorders. J Cell Mol Med 15:2025–2039. doi:10.1111/j.1582-4934.2011.01374.x Fan TJ, Han LH, Cong RS, Liang J (2005) Caspase family proteases and apoptosis. Acta Biochim Biophys Sin 37:719–727 Fischer JL, Lancia JK, Mathur A, Smith ML (2006) Selenium protection from DNA damage involves a Ref1/p53/Brca1 protein complex. Anticancer Res 26:899–904 Gao X, Xing H, Li S, Li J, Ying T, Xu S (2012) Selenium regulates gene expression of selenoprotein W in chicken
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gastrointestinal tract. Biol Trace Elem Res 145:181–188. doi:10.1007/s12011-011-9175-x Hammadi M et al (2013) Modulation of ER stress and apoptosis by endoplasmic reticulum calcium leak via translocon during unfolded protein response: involvement of GRP78. FASEB J 27:1600–1609. doi:10.1096/fj.12-218875 Hotamisligil GS (2010) Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 140:900–917. doi:10.1016/j.cell.2010.02.034 Irmak MB, Ince G, Ozturk M, Cetin-Atalay R (2003) Acquired tolerance of hepatocellular carcinoma cells to selenium deficiency: a selective survival mechanism? Cancer Res 63:6707–6715 Jergens A et al (2014) Bcl-2/caspase 3 mucosal imbalance favors T cell resistance to apoptosis in dogs with inflammatory bowel disease. Vet Immunol Immunopathol 158:167–174. doi:10.1016/j.vetimm.2014.01.004 Jiang C et al (2012) The role of the IRE1 pathway in PBDE-47induced toxicity in human neuroblastoma SH-SY5Y cells in vitro. Toxicol Lett 211:325–333. doi:10.1016/j.toxlet. 2012.04.009 Jimbo A et al (2003) ER stress induces caspase-8 activation, stimulating cytochrome c release and caspase-9 activation. Exp Cell Res 283:156–166 Kayanoki Y, Fujii J, Islam KN, Suzuki K, Kawata S, Matsuzawa Y, Taniguchi N (1996) The protective role of glutathione peroxidase in apoptosis induced by reactive oxygen species. J Biochem 119:817–822 Kim YS, Jhon DY, Lee KY (2004) Involvement of ROS and JNK1 in selenite-induced apoptosis in Chang liver cells. Exp Mol Med 36:157–164. doi:10.1038/emm.2004.22 Kim I, Xu W, Reed JC (2008) Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov 7:1013–1030. doi:10.1038/ nrd2755 Kitamura M, Hiramatsu N (2010) The oxidative stress: endoplasmic reticulum stress axis in cadmium toxicity. Biometals 23:941–950. doi:10.1007/s10534-010-9296-2 Ko WS et al (2005) Blood micronutrient, oxidative stress, and viral load in patients with chronic hepatitis C. World J Gastroenterol 11:4697–4702 Lee AS (2001) The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem Sci 26:504–510 Lee AS (2005) The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 35:373–381. doi:10.1016/j.ymeth.2004.10.010 Li HT, Feng L, Jiang WD, Liu Y, Jiang J, Li SH, Zhou XQ (2013) Oxidative stress parameters and anti-apoptotic response to hydroxyl radicals in fish erythrocytes: protective effects of glutamine, alanine, citrulline and proline. Aquat Toxicol 126:169–179. doi:10.1016/j.aquatox. 2012.11.005 Liang X, Xu K, Xu Y, Liu J, Qian X (2011) B1-induced caspaseindependent apoptosis in MCF-7 cells is mediated by down-regulation of Bcl-2 via p53 binding to P2 promoter TATA box. Toxicol Appl Pharmacol 256:52–61. doi:10. 1016/j.taap.2011.07.010 Liu XF, Zhang LM, Guan HN, Zhang ZW, Xu SW (2013) Effects of oxidative stress on apoptosis in manganese-induced testicular toxicity in cocks. Food Chem Toxicol 60:168–176. doi:10.1016/j.fct.2013.07.058
Biometals (2015) 28:255–265 Matsuda A, Kimura M, Itokawa Y (1998) Influence of selenium deficiency on vital functions in rats. Biol Trace Elem Res 61:287–301. doi:10.1007/BF02789089 Miao K, Zhang L, Yang S, Qian W, Zhang Z (2013) Intervention of selenium on apoptosis and Fas/FasL expressions in the liver of fluoride-exposed rats. Environ Toxicol Pharmacol 36:913–920. doi:10.1016/j.etap.2013.08.003 Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45 Qin P, Liu R (2013) Oxidative stress response of two fluoroquinolones with catalase and erythrocytes: a combined molecular and cellular study. J Hazard Mater 252–253:321–329. doi:10.1016/j.jhazmat.2013.03.006 Riedl SJ, Shi Y (2004) Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol 5:897–907. doi:10.1038/nrm1496 Ron D, Walter P (2007) Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8:519–529. doi:10.1038/nrm2199 Sano R, Reed JC (2013) ER stress-induced cell death mechanisms. Biochimica et biophysica acta 1833:3460–3470. doi:10.1016/j.bbamcr.2013.06.028 Schroder M, Kaufman RJ (2005) ER stress and the unfolded protein response. Mutat Res 569:29–63. doi:10.1016/j. mrfmmm.2004.06.056 Seong YA, Shin PG, Kim GD (2013) Anacardic acid induces mitochondrial-mediated apoptosis in the A549 human lung adenocarcinoma cells. Int J Oncol 42:1045–1051. doi:10. 3892/ijo.2013.1763 Seong YA, Shin PG, Yoon JS, Yadunandam AK, Kim GD (2014) Induction of the endoplasmic reticulum stress and autophagy in human lung carcinoma A549 cells by anacardic acid. Cell Biochem Biophys 68:369–377. doi:10. 1007/s12013-013-9717-2 Sharifi AM, Eslami H, Larijani B, Davoodi J (2009) Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells. Neurosci Lett 459:47–51. doi:10. 1016/j.neulet.2009.03.100 Sheng PF, Jiang Y, Zhang ZW, Zhang JL, Li S, Zhang ZQ, Xu SW (2014) The effect of Se-deficient diet on gene expression of inflammatory cytokines in chicken brain. Biometals 27:33–43. doi:10.1007/s10534-013-9682-7 Srivastava RK et al (2013) Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption. Toxicol Appl Pharmacol 272:879–887. doi:10.1016/j.taap.2013.08.004 Sun YY, Yin Y, Zhang JF, Yu HX, Wang XR, Wu JC, Xue YQ (2008) Hydroxyl radical generation and oxidative stress in Carassius auratus liver, exposed to pyrene. Ecotoxicol
265 Environ Saf 71:446–453. doi:10.1016/j.ecoenv.2007.12. 016 Sun B, Wang R, Li J, Jiang Z, Xu S (2011) Dietary selenium affects selenoprotein W gene expression in the liver of chicken. Biol Trace Elem Res 143:1516–1523. doi:10. 1007/s12011-011-8995-z Tagawa Y, Hiramatsu N, Kasai A, Hayakawa K, Okamura M, Yao J, Kitamura M (2008) Induction of apoptosis by cigarette smoke via ROS-dependent endoplasmic reticulum stress and CCAAT/enhancer-binding protein-homologous protein (CHOP). Free Radic Biol Med 45:50–59. doi:10. 1016/j.freeradbiomed.2008.03.003 Xu C, Bailly-Maitre B, Reed JC (2005) Endoplasmic reticulum stress: cell life and death decisions. J Clin Investig 115:2656–2664. doi:10.1172/JCI26373 Xu SW, Yao HD, Zhang J, Zhang ZW, Wang JT, Zhang JL, Jiang ZH (2013) The oxidative damage and disbalance of calcium homeostasis in brain of chicken induced by selenium deficiency. Biol Trace Elem Res 151:225–233. doi:10.1007/s12011-012-9552-0 Yao HD, Wu Q, Zhang ZW, Li S, Wang XL, Lei XG, Xu SW (2013a) Selenoprotein W serves as an antioxidant in chicken myoblasts. Biochim Biophys Acta 1830:3112–3120. doi:10.1016/j.bbagen.2013.01.007 Yao HD et al (2013b) Gene expression of endoplasmic reticulum resident selenoproteins correlates with apoptosis in various muscles of se-deficient chicks. J Nutr 143:613–619. doi:10.3945/jn.112.172395 Yokouchi M et al (2008) Involvement of selective reactive oxygen species upstream of proapoptotic branches of unfolded protein response. J Biol Chem 283:4252–4260. doi:10.1074/jbc.M705951200 Yu D, Li JL, Zhang JL, Gao XJ, Xu S (2011) Effects of dietary selenium on selenoprotein W gene expression in the chicken immune organs. Biol Trace Elem Res 144:678–687. doi:10.1007/s12011-011-9062-5 Zhang ZW, Zhang JL, Gao YH, Wang QH, Li S, Wang XL, Xu SW (2013) Effect of oxygen free radicals and nitric oxide on apoptosis of immune organ induced by selenium deficiency in chickens. Biometals 26:355–365. doi:10.1007/ s10534-013-9612-8 Zhang P, Sun Q, Zhao C, Ling S, Li Q, Chang YZ, Li Y (2014) HDAC4 protects cells from ER stress induced apoptosis through interaction with ATF4. Cell Signal 26:556–563. doi:10.1016/j.cellsig.2013.11.026 Zhou N, Xiao H, Li TK, Nur EKA, Liu LF (2003) DNA damagemediated apoptosis induced by selenium compounds. J Biol Chem 278:29532–29537. doi:10.1074/jbc. M301877200
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Roles of oxidative stress and endoplasmic reticulum stress in selenium deficiency-induced apoptosis in chicken liver Linlin Yao • Qiang Du • Haidong Yao Xi Chen • Ziwei Zhang • Shiwen Xu
•
Received: 16 September 2014 / Accepted: 26 December 2014 / Published online: 14 March 2015 Ó Springer Science+Business Media New York 2015
Abstract Oxidative stress and endoplasmic reticulum (ER) stress are involved in different types of stressinduced injuries. The aim of the present study was to evaluate the effect of Se deficiency on oxidative stress, ER stress and apoptosis in chicken livers. Chickens (1 day old, n = 180) were randomly divided into two groups: the L group [fed with a Se-deficient (Se 0.033 mg/kg) diet] and the control group [fed with a normal (Se 0.2 mg/kg) diet]. Factor-associated oxidative stress, catalase (CAT) activity, H2O2 production and the inhibition of hydroxyl radicals (OH) in the chicken liver were determined on days 15, 25, 35, 45, 55 and 65, respectively. In addition, ER stress-related genes (GRP78, GRP94, ATF4, ATF6 and IRE) and apoptosis-related genes (caspase3 and Bcl-2) were examined by fluorescence quantitative PCR or western blot analysis. Apoptosis levels were also measured using ultrastructural observations and the TdT-mediated dUTP nick end labeling assay. The results showed that CAT activity and OH inhibition were decreased and that H2O2 production was increased in the low-Se group, which demonstrated that oxidative stress occurred in the
L. Yao Q. Du H. Yao X. Chen Z. Zhang (&) S. Xu (&) Department of Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, People’s Republic of China e-mail: [email protected] S. Xu e-mail: [email protected]; [email protected]
chicken liver. The ER stress-related genes (GRP78, GRP94, ATF4, ATF6 and IRE) and the apoptosisrelated gene caspase3 were increased (p \ 0.05), while Bcl-2 was decreased (p \ 0.05) by Se deficiency. In addition, apoptosis and ER lesions were observed by ultrastructural observations of the chicken liver in the low-Se group. The level of apoptosis and the number of apoptotic cells increased with time. These results indicated that the oxidative-ER stress pathway participates in Se deficiency-induced apoptosis in the chicken liver. Keywords Selenium deficiency Oxidative stress Endoplasmic reticulum stress Apoptosis Chicken liver
Introduction Selenium (Se) deficiency is associated with numerous diseases. Se implements its biological function through incorporation into selenoproteins. In addition, Se deficiency caused injuries in several different chicken organs, including the brain (Sheng et al. 2014; Xu et al. 2013), muscles (Yao et al. 2013a, b), the gastrointestinal tract (Gao et al. 2012), the spleen (Yu et al. 2011) and the liver (Sun et al. 2011). The study of Fischer showed that the liver contains a high concentration of Se, which participates in many biological processes that range from cellular antioxidant defense to the protection and repair of DNA to apoptosis
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(Fischer et al. 2006). Se deficiency could induce liver necrosis in rats (Matsuda et al. 1998), hepatosis dietetica in pigs (Doyle et al. 2011), oxidative damage in the livers of patients with HCV-related CLD (Ko et al. 2005) and apoptosis in rat livers (Miao et al. 2013). Oxidative stress is described as an imbalance between reactive oxygen species (ROS) generation and antioxidant capacity, which triggers apoptosis through a variety of signaling pathways, such as the ER stress response (Kitamura and Hiramatsu 2010). ER stress is considered an early or initial response of cells to stress or damage (Doyle et al. 2011). The unfolded protein response (UPR) occurs in response to disturbed ER homeostasis and is induced to restore the protein folding capacity of ER through the accumulation of unfolded proteins in the ER lumen, which triggers both cell protective and cell death responses (Srivastava et al. 2013). GRP78 is a central regulator for ER stress due to its role as a major ER chaperone with anti-apoptotic properties as well as its ability to control the activation of transmembrane ER stress sensors (IRE1, PERK, and ATF6) through a binding-release mechanism (Hammadi et al. 2013; Lee 2005). ER stress is also related to the occurrence of apoptosis, in which CHOP, TRB3 (Zhang et al. 2014) and other apoptosis related genes (proapoptotic (caspase) and anti-apoptotic (Bcl-2 related family) are involved (Jergens et al. 2014). Previous studies showed that ER stress plays a key role in the regulation of apoptosis caused by a variety of toxic insults, including ROS chemicals and heavy metals. In chickens, Se deficiency causes oxidative damage in the brain (Xu et al. 2013) and immune tissues (Zhang et al. 2013). The study of Irmak showed that selenium deficiency-induced apoptosis in HCCderived cell lines was caused by oxidative stress (Irmak et al. 2003). In addition, Se deficiency could induce apoptosis in MDBK cells (Kayanoki et al. 1996) and NIH3T3 cells (Zhou et al. 2003). Therefore, diseases or damage related to Se deficiency may be mediated by oxidative-ER stress or apoptosis. However, the exact mechanism is still unclear. It has not been reported whether Se deficiency induced oxidative stress or ER stress or how these pathways regulate apoptosis in the chicken liver. In the present study, we examined factor-associated oxidative stress (CAT activity, H2O2 production and OH inhibition), ER stress (GRP78, GRP94, ATF4, ATF6 and IRE), and apoptosis (the apoptosis-associated markers caspase3
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and Bcl-2, ultrastructural observations and TUNEL assay) in chicken livers.
Materials and methods Animal care and experimental design All procedures used in the present study were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. Chickens (n = 180, 1-day-old, laying hens, Weiwei Co. Ltd., Harbin, China) were randomly divided into two groups (90 chickens per group). The chickens were maintained on either a Se-deficient granulated diet (L group, Se-deficient granulated diet including corn, soybean meal and wheat bran from Longjiang County, the Se deficiency region of Heilongjiang Province in China, and a blend of materials that did not add Se (Weiwei Co. Ltd., Harbin, China)) or the commercial granulated diet (Control group, Weiwei Co. Ltd., Harbin, China) for 15, 25, 35, 45, 55, or 65 days. The Se content of the Se-deficient granulated diet and the basal commercial granulated diet were analyzed by GB/T 13,883-2008 (PONY TEST Co., Beijing, China), which contained 0.033 and 0.200 mg/ kg Se (sodium selenite), respectively. The feed and water were supplied and libitum. Following euthanasia with sodium pentobarbital, livers (15, 25, 35, 45, 55 and 65 days old) were quickly collected and frozen immediately in liquid nitrogen and stored at -80 °C until required. Ultrastructural observations For electron microscopy, liver tissue specimens were fixed with 2.5 % glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 3 h at 4 °C, washed in the same buffer for 1 h at 4 °C and post fixed with 1 % osmium tetroxide in sodium phosphate buffer for 1 h at 4 °C. The tissues were then dehydrated in graded series of ethanol, 50, 70, 90 and 100 %, for 10 min each, and incubated twice in propylene oxide (100 % ethanol: 100 % propylene oxide = 1: 1 and 100 % propylene oxide for 10 min each at 4 °C). The tissue specimens were embedded in araldite. Ultrathin sections were stained with Mg-uranyl acetate and lead citrate for transmission electron microscope evaluation.
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Measurement of apoptosis by TUNEL assay TUNEL was carried out to analyze DNA fragmentation indicative of cellular apoptosis using the in situ cell death detection kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s protocol. Paraffin wax-embedded tissue sections were treated with proteinase K and the endogenous peroxidase activity was blocked with hydrogen peroxide. The sections were incubated at 37 °C with the terminal TdT nucleotide mixture for 1 h. Then, the reaction was stopped and the slides were rinsed with phosphate buffered saline (PBS). Nuclear labeling was developed with horseradish peroxidase and diaminobenzidine. Hematoxylin was used for counterstaining. The number of apoptotic cells in each slide from at least five different fields were analyzed using the Image-Pro Plus software (version 6.0 for Windows) with the aid of a microscope (BA400, Motic, China). The number of positive cells was averaged for statistical analysis. CAT activity, H2O2 production and OH inhibition assays Assays for CAT activity, H2O2 production and OH inhibition were performed at each time point using liver homogenates and specific kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s instructions. Western blot analysis Protein extracts were subjected to 15 % SDS-polyacrylamide gel electrophoresis under reducing conditions. The separated proteins were then transferred to nitrocellulose membranes for 2 h at 100 mA in a tank transfer apparatus containing Tris-glycine buffer and 20 % methanol. Membranes were blocked with 5 % skim milk for 24 h and incubated overnight with diluted primary antibodies against Bcl-2 (1:500, Santa Cruz Biotechnology, USA), caspase3 (1:100, Santa Cruz Biotechnology, USA) and GRP78 (1:1,000, polyclonal antibody produced by our lab) followed by a horseradish peroxidase (HRP)-conjugated secondary antibody against goat (Bcl-2) or rabbit (caspase3 and GRP78) IgG (1:1,000, Santa Cruz Biotechnology, USA). To verify equal loading of the samples, the membrane was incubated with a
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monoclonal b-actin antibody (1:1,000, Santa Cruz Biotechnology, USA), followed by a HRP-conjugated goat anti-mouse IgG (1:1,000) secondary antibody. The signal was detected with X-ray films (TransGen Biotech Co., Beijing, China). The optical density (OD) of each band was determined using an Image VCD gel imaging system, and the relative abundance of the Bcl2, caspase3 and GRP78 proteins were expressed as the ratios of OD of each of these proteins to that of b-actin. Quantitative real-time PCR analysis Total RNA was isolated from tissue samples using Trizol reagent, according to the manufacturer’s instructions (Invitrogen, Shanghai, China). The concentration and purity of the total RNA were determined spectrophotometrically at 260 and 280 nm using a GeneQuant 1300. The procedure of the reverse transcription was performed according to the manufacturer’s instructions (Invitrogen, Shanghai, China). Synthesized cDNA were stored at -20 °C for PCR. Oligo 7.0 Software was used to design specific primers for GRP78, GRP94, Bcl-2, caspase3, ATF4, ATF6, IRE and GADPH (a housekeeping gene used as an internal reference) based on known chicken sequences (Table 1). Primers were synthesized by Invitrogen Biotechnology Co. Ltd. in Shanghai, China. Quantitative real-time PCR was performed on an ABI PRISM 7500 Detection System (Applied Biosystems, Foster City, CA). Reactions were performed in a 20 lL reaction mixture containing 10 lL of the 29 SYBR Green I PCR Master Mix (TaKaRa, China), 2 lL cDNA, 0.4 lL of each primer (10 lM), 0.4 lL of the 509 ROX reference Dye II and 6.8 lL of PCRgrade water. The PCR procedure for GRP78, GRP94, Bcl-2, caspase3, ATF4, ATF6, IRE and GADPH consisted of 95 °C for 30 s followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 60 °C for 30 s. Dissociation curves were analyzed by Dissociation Curve 1.0 Software (Applied Biosystems) for each PCR reaction to detect and eliminate possible primerdimers and non-specific amplification. The relative abundance of each mRNA was calculated according to the method of Pfaffl (2001). Statistical analysis on all data was performed using GraphPad Prism 5.0 software, and all data were assessed using one-way ANOVA. Differences between the means were assessed using Tukey’s honestly significant
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Table 1 Gene-specific primers used in the real-time quantitative reverse transcription PCR Gene
Primer sequence
GRP 78
Forward 50 -GAATCGGCTAACACCAGAGGA-30 Reverse 50 -CGCATAGCTCTCCAGCTCATT-30
GRP94
Forward 50 -CAAAGACATGCTGAGGCGAGT-30 Reverse 50 -TCCACCTTTGCATCCAGGTCA-30
Bcl-2
Forward 50 -ATCGTCGCCTTCTTCGAGTT-30 Reverse 50 -ATCCCATCCTCCGTTGTCCT-30
Caspase3
Forward 50 -CATCT GCATCC GTGCCTGA-30 Reverse 50 -CTCTCGG CTGTGGTGGTGAA-30
ATF4
Forward 50 -GAATCGGCTAACACCAGAGGA-30 Reverse 50 -CGCATAGCTCTCCAGCTCATT-30
ATF6
Forward 50 - CGTCGTCTGAACCACTTACTGA-30 Reverse 50 -CCTTCTTTCCTAACAGCCACAC-30
IRE
Forward 50 -CTACAGGTCGCTCCTCACATC-30 Reverse 50 -ATCAGTCCTTCTGCTCCCATCT-30
GADPH
had brown stained nuclei. Livers from the L group had an increased number of apoptotic cells compared to the control group, which further demonstrated that Se deficiency promoted apoptosis in chicken livers (Fig. 2).The effects of Se deficiency on the percent of apoptotic cells in liver were shown in Fig. 3. The percent of apoptotic cells in liver was increased (p \ 0.05) in the L group compared to the control group from 15 to 65 days of age. A significant increase in the percent of apoptotic cells in the liver was observed over time for chickens that were fed a Sedeficient basal diet.
0
0
Forward 5 -AGAACATCATCCCAGCGT-3 Reverse 50 -AGCCTTCACTACCCTCTTG-30
difference test for post hoc multiple comparisons. All data were expressed as the mean ± SD, where p \ 0.05 was considered a statistically significant difference.
Changes in CAT activity, H2O2 production and OH inhibition The effects of Se deficiency on CAT activity and OH inhibition in liver were shown in Fig. 4a, c respectively. These parameters were decreased (p \ 0.05) in the L groups as compared to the control group from 15 to 65 days of age. H2O2 production increased (p \ 0.05) from 25 to 65 days of age (Fig. 4b). Effects of Se deficiency on the expression of ER stress-related genes (GRP78, GRP94, ATF4, ATF6 and IRE) in chicken livers
Results Ultrastructural changes No ultrastructural changes were observed in the liver of control group (Fig. 1a). The mitochondria from the liver of the L group were vacuolated and the cristae (black arrows) had degenerated. The apoptotic cells showed typical condensed nuclei with horseshoe-like or crescent-shaped cytoplasmic organelles of an inconspicuous structure (red arrows) accompanied by nucleus shrinkage (blue arrows). The nuclei and organelles of some cells were unclear (Fig. 1b). The mitochondria from the liver of the L group were surrounded by fractured ER (black arrows) (Fig. 1c). Their rough ER was extended (black arrows) (Fig. 1d). Measurement of cellular apoptosis using the TUNEL assay We performed the TUNEL assay in chicken livers to confirm the occurrence of apoptosis. Apoptotic cells
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The effects of Se deficiency on the levels of GRP78, GRP94, ATF4, ATF6 and IRE mRNA were shown in Fig. 7a–e, respectively. They were increased (p \ 0.05) in the L groups compared to the control group over time. Notably, the levels of GRP78 and GRP94 mRNA in the L group increased by 21.48 and 22.36 fold, respectively, compared to the control group at 65 days of age. Moreover, the relative levels of GRP78 protein were shown in Figs. 5 and 6a and were also increased (p \ 0.05) in the L group compared to the control groups from 15 to 65 days of age. Effects of Se deficiency on the expression of the apoptosis-related genes (Bcl-2 and caspase3) in chicken livers The effects of Se deficiency on the levels of Bcl-2 and caspase3 mRNA were shown in Fig. 7f, g, while the levels of each protein were shown in Figs. 5 and 6b, c, respectively. Bcl-2 was decreased (p \ 0.05) and
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Fig. 1 Effects of Se deficiency on the ultrastructural changes in liver of chickens from the a control group and b–d L group
caspase3 was increased (p \ 0.05) by Se deficiency at both the mRNA and protein levels.
Discussion Se deficiency induced liver injury, apoptosis and necrosis in different types of animals; however, few reports exist for chicken livers, and the exact mechanism of Se deficiency on liver apoptosis remains unclear. In the present study, evident apoptotic characteristics and an increased rate of apoptosis were observed in the liver of Se-deficient chicken compared with the control group over time. The experimental groups were accompanied by oxidative stress and ER stress, which launched the oxidative-ER apoptosis pathway.
Se deficiency has been shown to induce the oxidative stress in different tissues. We previously showed that Se deficiency could cause oxidative damage in chicken brain (Xu et al. 2013) and immune tissues (Zhang et al. 2013). CAT activity, H2O2 production and OH inhibition are the main biological markers of oxidative stress in tissues and organs. CAT is a heme-containing redox enzyme and serves to protect the body from the toxic damage of H2O2 by catalyzing its decomposition into molecular oxygen and water without the production of free radicals, such as OH. According to reports,CAT is closely associated with an increased risk of oxidative stress; ciprofloxacin and enrofloxacin significantly decreased CAT activity compared to the control group (Qin and Liu 2013). Li proved that OH induces apoptosis and oxidative damage in carp erythrocytes (Li et al. 2013).
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Fig. 2 Effects of Se deficiency on the TUNEL assay in chicken livers. a Central veins of the liver in the control group. b Central veins of the liver in the L group. c Portal area of the liver in the control group. d Portal area of the liver in the L group
Fig. 3 Effects of Se deficiency on the apoptotic rate chicken livers. The asterisk indicated that there were significant differences (p \ 0.05) between the control group and the L group at the same time point. Each value represented the mean ± SD of five individuals
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The study of Sun found that the OH was generated in Carassius auratus liver that was exposed to pyrene. The OH production showed a significant increase compared with the control groups (Sun et al. 2008). Liu pointed that the inhibition of OH in the testicles was dose- and time-dependent. There was a daily decrease in the inhibition of OH activity for all experimental groups compared with that of the control group. These results clearly showed that oxidative stress and damage occurred in response to Mn (Liu et al. 2013). In the present study, the activity of CAT was decreased, H2O2 production was increased, and the OH inhibition was decreased in the Se-deficient group compared to the control group. Therefore, similar to prior studies, the present study showed that Se deficiency induced oxidative stress in chicken liver. However, the occurrence of oxidative stress induced continuous ER stress through interfering with
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Fig. 4 Effects of Se deficiency on the CAT activity, H2O2 production and OH inhibition chicken livers. a Effects of Se deficiency on CAT activity. b Effects of Se deficiency on H2O2 production. c Effects of Se deficiency on inhibiting OH ability.
Fig. 5 Effects of Se deficiency on the levels of the GRP78, Bcl2 and caspase3 proteins. The notations 15C, 25C, 35C, 45C, 55C and 65C indicated the control groups from 15 days of age, 25 days of age, 35 days of age, 45 days of age, 55 days of age and 65 days of age, respectively. The notations 15L, 25L, 35L, 45L, 55L and 65L respectively indicated the L groups from 15 days of age, 25 days of age, 35 days of age, 45 days of age, 55 days of age and 65 days of age, respectively
the oxidation of the internal environment of the ER. The latest research also showed for some of the factors involved in the induction of apoptosis, such as tumor necrosis factor (Burton et al. 2009), cigarettes (Tagawa et al. 2008) and cadmium intoxication (Yokouchi et al. 2008), the ER stress response may be downstream of oxidative stress.
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The asterisk indicated that there were significant differences (p \ 0.05) between the control group and the L group at the same time point. Each value represented the mean ± SD of five individuals
When ER stress occurred, chaperone proteins, such as GRP78 and GRP94, are recruited to support the folding of new proteins (Hotamisligil 2010). Therefore, the UPR can enhance the capabilities of the ER to process paraproteins and up-regulate the expression of ER chaperones GRP78 and GRP94 (Lee 2001). A previous study showed that exposure to a pyrrolidine dithiocarbamate (PDTC)/Cu complex could induce cytotoxicity and apoptosis in alveolar epithelial cells via the mitochondria- and ER-stressrelated signaling pathways; during these processes, the expression of ER stress-associated signaling molecules including GRP78, GRP94 and ATF4 were increased by the PDTC/Cu complex (Chen et al. 2010). In our study, the expression GRP78 and GRP94 were significantly elevated in the liver of the L group over time. The up-regulated expression of these proteins clearly indicated that Se deficiency could lead to ER stress. In addition, ER lesions were observed by ultrastructural observations in chicken liver in the low-Se group.
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Fig. 6 Effects of Se deficiency on the levels of the GRP78, Bcl2 and caspase3 proteins in chicken livers. a Effects of Se deficiency on the relative level of GRP78 protein in liver. b Effects of Se deficiency on the relative level of Bcl-2 protein in liver. c Effects of Se deficiency on the relative level of caspase3
protein in liver. The results were from at least three independent experiments. The asterisk indicated that there were significant differences (p \ 0.05) between the control group and the L group at the same time point. Each value represented the mean ± SD
Moreover, upon ER stress, P19 embryonal carcinoma (EC) cells activated various caspases, including caspase3, and exhibited extensive DNA fragmentation (Jimbo et al. 2003). Caspases are key regulators of cell apoptosis,and are involved in ER stressed-induced apoptosis (Fan et al. 2005; Riedl and Shi 2004). Caspase3 activity was significantly increased in treated PC12 cells compared to controls during high glucose-induced apoptosis (Sharifi et al. 2009). On the other hand, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to a decrease in mRNA levels (Liang et al. 2011). The study of Chen also found that Bcl-2 mRNA and protein expressions were decreased in lung epithelial cells during the process of apoptosis induced by ER stress (Chen et al. 2010). Studies have also found that Se deficiency causes cell cycle arrest and increased apoptotic activity (Kim et al. 2004). In present study, both caspase3 mRNA and protein increased and Bcl-2 mRNA and protein levels decreased in the experimental group. In addition, apoptosis was observed by ultrastructural observations and TUNEL assay in chicken liver. These results showed that ER stress is related to the occurrence of apoptosis.
It has been shown that ER stress occurred by several pathways including IRE1, PERK, and ATF6 (Ron and Walter 2007; Sano and Reed 2013; Xu et al. 2005). Among them, the PERK pathway enhances translation of mRNAs, including ATF4 (Armstrong et al. 2010). In a previous study, anacardic acid (6-pentadecylsalicylic acid, AA), which induced apoptosis in pituitary adenoma and lung adenocarcinoma cells through ER stress, not only significantly increased the expression of GRP78, but also increased the expression of ATF4 in both HepG2 and U266 cells (Seong et al. 2013, 2014). IRE1 is derived from the activation of IRE. Considerable evidence has demonstrated a connection between apoptosis and the ER stress and IRE1 pathways (Jiang et al. 2012; Kim et al. 2008). The IRE1 pathway, the most evolutionarily conserved pathway of the three UPR pathways, has a proapoptotic function that involves the activation of GRP78 and IRE1 (Schroder and Kaufman 2005). ATF6 is an ER stress-regulated transmembrane transcription factor that activates the transcription of ER molecular chaperones. In our results, the levels of ATF4, ATF6 and IRE mRNA were increased in the L group compared to the control group. In a word, these
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Fig. 7 Effects of Se deficiency on the levels of GRP78, GRP94, ATF4, ATF6, IRE, Bcl-2 and caspase3 mRNA in chicken livers. a Effects of Se deficiency on the level of GRP78 mRNA in liver. b Effects of Se deficiency on the level of GRP94 mRNA in liver. c Effects of Se deficiency on the level of ATF4 mRNA in liver. d Effects of Se deficiency on the level of ATF6 mRNA in liver. e Effects of Se deficiency on the level of IRE mRNA in liver. f Effects of Se deficiency on the level of Bcl-2 mRNA in liver. g Effects of Se deficiency on the level of caspase3 mRNA in liver. The asterisk indicated that there were significant differences (p \ 0.05) between the control group and the L group at the same time point. Each value represented the mean ± SD of five individuals
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results illustrated that Se deficiency-induced ER stress may be involved in the PERK, ATF6 and IRE1 pathways.
Conclusions The present study showed that Se deficiency led to the occurrence of oxidative stress, ER stress and apoptosis in chicken livers. Moreover, Se deficiency-induced ER stress may be involved in the PERK, ATF6 and IRE1 pathways. Therefore, the oxidative-ER stress pathway participates in the process of Se deficiency-induced apoptosis in the chicken liver. Acknowledgments This study was supported by the National Natural Science Foundation of China (31272626); the International (Regional) Cooperation and Exchange Projects of the National Natural Science Foundation of China (31320103920) and the Study Abroad Foundation of Heilongjiang Province (LC201031). Conflict of interest conflicts of interest.
The authors declare that there are no
References Armstrong JL, Flockhart R, Veal GJ, Lovat PE, Redfern CP (2010) Regulation of endoplasmic reticulum stress-induced cell death by ATF4 in neuroectodermal tumor cells. J Biol Chem 285:6091–6100. doi:10.1074/jbc.M109. 014092 Burton GJ, Yung HW, Cindrova-Davies T, Charnock-Jones DS (2009) Placental endoplasmic reticulum stress and oxidative stress in the pathophysiology of unexplained intrauterine growth restriction and early onset preeclampsia. Placenta 30(Suppl A):S43–S48. doi:10.1016/j.placenta. 2008.11.003 Chen YW et al (2010) Pyrrolidine dithiocarbamate (PDTC)/Cu complex induces lung epithelial cell apoptosis through mitochondria and ER-stress pathways. Toxicol Lett 199:333–340. doi:10.1016/j.toxlet.2010.09.016 Doyle KM, Kennedy D, Gorman AM, Gupta S, Healy SJ, Samali A (2011) Unfolded proteins and endoplasmic reticulum stress in neurodegenerative disorders. J Cell Mol Med 15:2025–2039. doi:10.1111/j.1582-4934.2011.01374.x Fan TJ, Han LH, Cong RS, Liang J (2005) Caspase family proteases and apoptosis. Acta Biochim Biophys Sin 37:719–727 Fischer JL, Lancia JK, Mathur A, Smith ML (2006) Selenium protection from DNA damage involves a Ref1/p53/Brca1 protein complex. Anticancer Res 26:899–904 Gao X, Xing H, Li S, Li J, Ying T, Xu S (2012) Selenium regulates gene expression of selenoprotein W in chicken
123
gastrointestinal tract. Biol Trace Elem Res 145:181–188. doi:10.1007/s12011-011-9175-x Hammadi M et al (2013) Modulation of ER stress and apoptosis by endoplasmic reticulum calcium leak via translocon during unfolded protein response: involvement of GRP78. FASEB J 27:1600–1609. doi:10.1096/fj.12-218875 Hotamisligil GS (2010) Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 140:900–917. doi:10.1016/j.cell.2010.02.034 Irmak MB, Ince G, Ozturk M, Cetin-Atalay R (2003) Acquired tolerance of hepatocellular carcinoma cells to selenium deficiency: a selective survival mechanism? Cancer Res 63:6707–6715 Jergens A et al (2014) Bcl-2/caspase 3 mucosal imbalance favors T cell resistance to apoptosis in dogs with inflammatory bowel disease. Vet Immunol Immunopathol 158:167–174. doi:10.1016/j.vetimm.2014.01.004 Jiang C et al (2012) The role of the IRE1 pathway in PBDE-47induced toxicity in human neuroblastoma SH-SY5Y cells in vitro. Toxicol Lett 211:325–333. doi:10.1016/j.toxlet. 2012.04.009 Jimbo A et al (2003) ER stress induces caspase-8 activation, stimulating cytochrome c release and caspase-9 activation. Exp Cell Res 283:156–166 Kayanoki Y, Fujii J, Islam KN, Suzuki K, Kawata S, Matsuzawa Y, Taniguchi N (1996) The protective role of glutathione peroxidase in apoptosis induced by reactive oxygen species. J Biochem 119:817–822 Kim YS, Jhon DY, Lee KY (2004) Involvement of ROS and JNK1 in selenite-induced apoptosis in Chang liver cells. Exp Mol Med 36:157–164. doi:10.1038/emm.2004.22 Kim I, Xu W, Reed JC (2008) Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov 7:1013–1030. doi:10.1038/ nrd2755 Kitamura M, Hiramatsu N (2010) The oxidative stress: endoplasmic reticulum stress axis in cadmium toxicity. Biometals 23:941–950. doi:10.1007/s10534-010-9296-2 Ko WS et al (2005) Blood micronutrient, oxidative stress, and viral load in patients with chronic hepatitis C. World J Gastroenterol 11:4697–4702 Lee AS (2001) The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem Sci 26:504–510 Lee AS (2005) The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 35:373–381. doi:10.1016/j.ymeth.2004.10.010 Li HT, Feng L, Jiang WD, Liu Y, Jiang J, Li SH, Zhou XQ (2013) Oxidative stress parameters and anti-apoptotic response to hydroxyl radicals in fish erythrocytes: protective effects of glutamine, alanine, citrulline and proline. Aquat Toxicol 126:169–179. doi:10.1016/j.aquatox. 2012.11.005 Liang X, Xu K, Xu Y, Liu J, Qian X (2011) B1-induced caspaseindependent apoptosis in MCF-7 cells is mediated by down-regulation of Bcl-2 via p53 binding to P2 promoter TATA box. Toxicol Appl Pharmacol 256:52–61. doi:10. 1016/j.taap.2011.07.010 Liu XF, Zhang LM, Guan HN, Zhang ZW, Xu SW (2013) Effects of oxidative stress on apoptosis in manganese-induced testicular toxicity in cocks. Food Chem Toxicol 60:168–176. doi:10.1016/j.fct.2013.07.058
Biometals (2015) 28:255–265 Matsuda A, Kimura M, Itokawa Y (1998) Influence of selenium deficiency on vital functions in rats. Biol Trace Elem Res 61:287–301. doi:10.1007/BF02789089 Miao K, Zhang L, Yang S, Qian W, Zhang Z (2013) Intervention of selenium on apoptosis and Fas/FasL expressions in the liver of fluoride-exposed rats. Environ Toxicol Pharmacol 36:913–920. doi:10.1016/j.etap.2013.08.003 Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45 Qin P, Liu R (2013) Oxidative stress response of two fluoroquinolones with catalase and erythrocytes: a combined molecular and cellular study. J Hazard Mater 252–253:321–329. doi:10.1016/j.jhazmat.2013.03.006 Riedl SJ, Shi Y (2004) Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol 5:897–907. doi:10.1038/nrm1496 Ron D, Walter P (2007) Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8:519–529. doi:10.1038/nrm2199 Sano R, Reed JC (2013) ER stress-induced cell death mechanisms. Biochimica et biophysica acta 1833:3460–3470. doi:10.1016/j.bbamcr.2013.06.028 Schroder M, Kaufman RJ (2005) ER stress and the unfolded protein response. Mutat Res 569:29–63. doi:10.1016/j. mrfmmm.2004.06.056 Seong YA, Shin PG, Kim GD (2013) Anacardic acid induces mitochondrial-mediated apoptosis in the A549 human lung adenocarcinoma cells. Int J Oncol 42:1045–1051. doi:10. 3892/ijo.2013.1763 Seong YA, Shin PG, Yoon JS, Yadunandam AK, Kim GD (2014) Induction of the endoplasmic reticulum stress and autophagy in human lung carcinoma A549 cells by anacardic acid. Cell Biochem Biophys 68:369–377. doi:10. 1007/s12013-013-9717-2 Sharifi AM, Eslami H, Larijani B, Davoodi J (2009) Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells. Neurosci Lett 459:47–51. doi:10. 1016/j.neulet.2009.03.100 Sheng PF, Jiang Y, Zhang ZW, Zhang JL, Li S, Zhang ZQ, Xu SW (2014) The effect of Se-deficient diet on gene expression of inflammatory cytokines in chicken brain. Biometals 27:33–43. doi:10.1007/s10534-013-9682-7 Srivastava RK et al (2013) Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption. Toxicol Appl Pharmacol 272:879–887. doi:10.1016/j.taap.2013.08.004 Sun YY, Yin Y, Zhang JF, Yu HX, Wang XR, Wu JC, Xue YQ (2008) Hydroxyl radical generation and oxidative stress in Carassius auratus liver, exposed to pyrene. Ecotoxicol
265 Environ Saf 71:446–453. doi:10.1016/j.ecoenv.2007.12. 016 Sun B, Wang R, Li J, Jiang Z, Xu S (2011) Dietary selenium affects selenoprotein W gene expression in the liver of chicken. Biol Trace Elem Res 143:1516–1523. doi:10. 1007/s12011-011-8995-z Tagawa Y, Hiramatsu N, Kasai A, Hayakawa K, Okamura M, Yao J, Kitamura M (2008) Induction of apoptosis by cigarette smoke via ROS-dependent endoplasmic reticulum stress and CCAAT/enhancer-binding protein-homologous protein (CHOP). Free Radic Biol Med 45:50–59. doi:10. 1016/j.freeradbiomed.2008.03.003 Xu C, Bailly-Maitre B, Reed JC (2005) Endoplasmic reticulum stress: cell life and death decisions. J Clin Investig 115:2656–2664. doi:10.1172/JCI26373 Xu SW, Yao HD, Zhang J, Zhang ZW, Wang JT, Zhang JL, Jiang ZH (2013) The oxidative damage and disbalance of calcium homeostasis in brain of chicken induced by selenium deficiency. Biol Trace Elem Res 151:225–233. doi:10.1007/s12011-012-9552-0 Yao HD, Wu Q, Zhang ZW, Li S, Wang XL, Lei XG, Xu SW (2013a) Selenoprotein W serves as an antioxidant in chicken myoblasts. Biochim Biophys Acta 1830:3112–3120. doi:10.1016/j.bbagen.2013.01.007 Yao HD et al (2013b) Gene expression of endoplasmic reticulum resident selenoproteins correlates with apoptosis in various muscles of se-deficient chicks. J Nutr 143:613–619. doi:10.3945/jn.112.172395 Yokouchi M et al (2008) Involvement of selective reactive oxygen species upstream of proapoptotic branches of unfolded protein response. J Biol Chem 283:4252–4260. doi:10.1074/jbc.M705951200 Yu D, Li JL, Zhang JL, Gao XJ, Xu S (2011) Effects of dietary selenium on selenoprotein W gene expression in the chicken immune organs. Biol Trace Elem Res 144:678–687. doi:10.1007/s12011-011-9062-5 Zhang ZW, Zhang JL, Gao YH, Wang QH, Li S, Wang XL, Xu SW (2013) Effect of oxygen free radicals and nitric oxide on apoptosis of immune organ induced by selenium deficiency in chickens. Biometals 26:355–365. doi:10.1007/ s10534-013-9612-8 Zhang P, Sun Q, Zhao C, Ling S, Li Q, Chang YZ, Li Y (2014) HDAC4 protects cells from ER stress induced apoptosis through interaction with ATF4. Cell Signal 26:556–563. doi:10.1016/j.cellsig.2013.11.026 Zhou N, Xiao H, Li TK, Nur EKA, Liu LF (2003) DNA damagemediated apoptosis induced by selenium compounds. J Biol Chem 278:29532–29537. doi:10.1074/jbc. M301877200
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