Accepted Manuscript Title: Deoxynivalenol induces apoptosis in chicken splenic lymphocytes via the reactive oxygen species-mediated mitochondrial pathway Author: Zhihua Ren Yachao Wang Huidan Deng Youtian Deng Junliang Deng Zhicai Zuo Ya Wang Xi Peng Hengmin Cui Liuhong Shen PII: DOI: Reference:
S1382-6689(14)00298-1 http://dx.doi.org/doi:10.1016/j.etap.2014.11.028 ENVTOX 2145
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
3-6-2014 11-11-2014 15-11-2014
Please cite this article as: Ren, Z., Wang, Y., Deng, H., Deng, Y., Deng, J., Zuo, Z., Wang, Y., Peng, X., Cui, H., Shen, L.,Deoxynivalenol induces apoptosis in chicken splenic lymphocytes via the reactive oxygen speciesmediated mitochondrial pathway, Environmental Toxicology and Pharmacology (2014), http://dx.doi.org/10.1016/j.etap.2014.11.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DON inhibited the growth of splenic lymphocytes in a dose-dependent manner. Cytotoxicity was assessed with the CCK-8 bioassay. DON treatment induced ROS accumulation in splenic lymphocytes. Apoptosis was correlated with inappropriate of p53, Bax, Bak-1, Bcl-2, and caspase-3 concentrations. 5. Apoptosis was correlated with disordered of p53, Bax, Bak-1, Bcl-2, and caspase-3 expression.
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Deoxynivalenol induces apoptosis in chicken splenic lymphocytes via the reactive oxygen species-mediated mitochondrial pathway Zhihua Ren1, Yachao Wang1, 2, Huidan Deng1, Youtian Deng1, Junliang Deng1*, Zhicai Zuo1, Ya
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Wang1, Xi Peng1, Hengmin Cui1, Liuhong Shen1
1 College of Veterinary Medicine, Sichuan Agricultural University; Sichuan Province Key
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Laboratory of Animal Disease ＆ Human Health; Key Laboratory of Environmental Hazard and Human Health of Sichuan Province, Ya’an, 625014, China;
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2 School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, 621010, China.
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Email address:
Zhihua Ren (Doctor, associate Professor): [email protected]
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Yachao Wang (Doctor, Lecturer): [email protected] Huidan Deng (Master): [email protected]
Youtian Deng (Undergraduate):[email protected]
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corresponding author (Doctor, Professor) : Junliang Deng: [email protected]
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Zhicai Zuo (Doctor, Professor): [email protected] Ya Wang (Master, experimentalist): [email protected]
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Xi Peng (Doctor, Professor): [email protected] Hengmin Cui (Doctor, Professor): [email protected] Liuhong Shen (Doctor, associate Professor): [email protected]
E-mail address of corresponding author: [email protected] (J. L. Deng). Telephone: +86 835 2885313; fax:+86 835 2885302.
Postal address of corresponding author: College of Veterinary Medicine, Sichuan Agricultural University, Ya’an, Sichuan 625014, China **Z. H. Ren and Y. C. Wang contributed equally to this work and should be considered co-first authors.
The authors confirm that this manuscript has not been published elsewhere and is not under
Page 2 of 25
consideration by another journal. All authors have approved the manuscript and agree with submission to the Toxicology in Vitro. The authors have no conflicts of interest to declare.
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ABSTRACT We investigated the immunotoxicity and cytotoxicity of deoxynivalenol (DON), a
mycotoxin, and the mechanism by which it induces apoptosis. Chicken splenic
cr
lymphocytes treated with 0-50 µg/mL DON for 48 h inhibited growth of splenic
us
lymphocytes in a dose-dependent manner, as revealed by the Cell Counting Kit-8 (CCK-8) bioassay. Annexin V-fluorescein isothiocyanate staining indicated that the number of apoptotic and necrotic cells were significantly higher compared with the
an
control (P < 0.01). DON treatment induced ROS accumulation, resulting in reduced mitochondrial transmembrane potential, as detected by flow cytometry and 2’,
M
7’-dichlorofluorescein acetate and rhodamine 123 labeling, respectively. Enzyme linked immunosorbent assays revealed that the concentrations of p53, Bax, Bak-1, and
d
Caspase-3 increased with increasing DON concentration (P < 0.05 or P < 0.01),
te
whereas the concentrations of Bcl-2 decreased (P < 0.01) compared with the control. These data suggest that DON induces apoptosis in splenic lymphocytes via a
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ROS-mediated mitochondrial pathway. Keywords: Deoxynivalenol; apoptosis; mitochondria; reactive oxygen species
Abbreviations:
CCK-8, Cell Counting Kit-8; ConA, concanavalin A; DON, Deoxynivalenol; ELISA, enzyme linked immunosor bent assay; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HO, hydroxyl radicals; H2O2, hydrogen peroxide; METC1, mouse
thymic epithelial cell line 1; MMP ΔΨm, mitochondrial membrane potential; O2−, superoxide; OD, optical density; PBS, phosphate-buffered saline; PI, propidium iodide; ROS, reactive oxygen species; SD, standard deviation;
Page 3 of 25
1.
INTRODUCTION Mycotoxins are secondary metabolites of molds, which contaminate a wide range
of crop plants and fruits before or after harvest. Aflatoxins, such as deoxynivalenol
ip t
(DON), ochratoxin A, fumonisin, zearalenone, patulin and T-2 toxin, are among the most common mycotoxins. They are characteristically stable under changing
cr
environmental conditions and cause various toxic effects in experimental animals, livestock and humans (Chen et al., 2008).
us
DON, also known as vomitoxin, is a mycotoxin classified as a type B trichothecene, which is produced mainly by Fusarium graminearum and F. culmorum.
an
DON is present at toxicologically relevant concentrations in cereals and grains worldwide. Furthermore, DON is resistant to milling, processing and heating, and
M
therefore readily enters both the human and animal food chains (Jackson et al., 1999; Rotter et al., 1996). To date, DON has been shown to exhibit toxic effects in humans
d
and all animal species, impairing growth in experimental animals and inducing dysregulation of the immune system (Accensi et al., 2006; Pestka et al., 2005; Pinton
te
et al., 2008). Depending on the dose and frequency of exposure, DON can be either
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immunosuppressive or immunostimulatory. At the cellular level, DON interacts with peptidyl transferase in the 60S ribosomal subunit and triggers ribotoxic stress (Pestka, 2008). The multiple cellular effects of trichothecenes on eukaryotic cells have been reviewed elsewhere (Rocha et al., 2005). In summary, trichothecenes inhibit protein, DNA and RNA synthesis, and mitochondrial function, and they also affect cell division, membrane integrity, and induce apoptosis. Lymphoid cells and fibroblasts are the most DON-sensitive cell types. One of the most important effects of the trichothecene mycotoxins is impairment of immune function. However, most in vivo immunotoxicity studies have been conducted in the mouse or pig (Goyarts et al, 2006; Tiemann et al, 2006). In contrast, numerous DON cytotoxicity studies have been conducted in humans, laboratory animals (mice, rats, and guinea pigs) and domestic pigs (Pestka et al., 2005; Calvert et al., 2005; Cetin and Bullerman, 2005; Fornelli et al., 2004; Minervini et al., 2004;
Page 4 of 25
Pestka, 2007). Many studies have suggested that the DON-induced production of free radicals may be one of the mechanisms that cause damage to the cellular membrane and to DNA. Thus, oxidative stress is regarded as an important factor in DON-induced toxicity (Rizzo et al., 1994). Recent studies have demonstrated that
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DON is able to decrease cell viability and cause damage to the cellular membrane,
chromosomes, or DNA. It is also able to induce lipid peroxidation and raise the levels
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of 8-OHdG and ROS in human peripheral blood lymphocytes (Yang et al., 2014). However, to our knowledge, studies on DON-induced apoptosis in chicken cells are
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limited. The present study evaluates the effects of DON in primary chicken splenic
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lymphocytes and explores the mechanism of apoptosis induction.
2. 2. MATERIALS AND METHODS 2.1 Chemicals
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DON, RPMI 1640 medium, and 2’,7’-dichlorofluorescein diacetate (DCFH-DA) and rhodamine 123 were purchased from Sigma-Aldrich (St Louis, MO, USA).
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Histopaque 1077 and the Cell Counting Kit-8 (CCK-8) were obtained from Dojindo Laboratories (Tokyo, Japan). The annexin V-fluorescein isothiocyanate (FITC)
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Apoptosis Detection kit was purchased from BD Pharmingen (Lexington, KY, USA). The chicken Bc1-2, p53, Bax, Bak-1, and Caspase-3 ELISA kits were obtained from
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Qiyi Biological Technology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) was purchased from Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). Trizol reagent and the DNA extraction kit were purchased from Invitrogen Biotechnology Co., Ltd. (Shanghai, China). The SYBR PremixScript RT-PCR Kit II was purchased from Takara (Shiga, Japan). All other reagents used were of analytical grade.
2.2 Cell Isolation and Culture All procedures in this study were approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University. One hundred and eighty 1-day-old healthy male Isa Brown chickens were obtained from a comercial rearing farm (Jilin poultry farm, Jilin province) at day of hatching. One-day old chickens were placed in
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large pens with wood shavings and were reared with lighting regimen 23 h light and 1 h dark. The initial room temperature of 32-33 °C was reduced weekly by 1 °C to a final temperature of 28 °C. The relative humidity was within a range 50-60%. The chickens provided with water as well as feed ad libitum. And the chickens were fed
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only a basic commercial diet. Nutritional requirements were adequate according to
National Research Council (NRC, 1994) and the Chinese Feeding Standard of
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Chickens (NY/T33-2004). At 8 weeks of age, chickens in good health were
anaesthetized with an intramuscular injection of 846 anesthetic mixture (haloperidol,
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dihydroetorphine, and 2, 4 - dimethylaniline thiazole) (Duan et al, 2005) using doses 0.8 mL/kg body wright, respectively. After laparotomy, spleen samples of chickens
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were removed from the experimental animal.
The spleens were ground on ice and teased through a 200-mesh cell strainer into a Petri dish containing phosphate-buffered saline (PBS). The cell suspension was
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overlaid onto Histopaque 1077 and centrifuged at 400 × g for 15 min at room temperature. Lymphocytes at the interface were collected, washed twice with PBS by
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centrifugation at 250 × g for 5 min at room temperature, and then suspended in RPMI-1640 medium (without phenol red) supplemented with 10% (v/v) FBS, 100
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U/mL penicillin, and 100 U/mL streptomycin. More than 95% of cells were viable
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based on trypan blue dye exclusion.
To monitor various parameters (excluding the CCK-8 bioassay), splenic
lymphocytes were cultured in six-well culture plates (6 × 106 cells/mL) in
quintuplicate, stimulated with 12.5 µg/mL concanavalin A (ConA) to induce cell proliferation and treated with 0.0, 0.2, 0.8, 3.2, 12.5 or 50.0 µg/mL DON at 41.5 °C in
a humidified atmosphere with 5% (v/v) CO2 for 48 h (Okamura et al., 2004). After 48 h of incubation, the supernatants were collected for Bc1-2, p53, Bax, Bak-1, and Caspase-3 analysis. Cells were collected to analyze the rate of apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP ΔΨm) and for DNA and RNA isolation. Both the supernatants and the cells were frozen at
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80 °C until use.
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−
2.3 CCK-8 Bioassay
Cell viability was quantified using the CCK-8 bioassay as described elsewhere
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(Miyamoto et al., 2002). Splenic lymphocytes were seeded into 96-well culture plates
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(6 × 105/well) and treated with 0–50 µg/mL DON and 12.5 µg/ml ConA for 48 h at 41.5 °C with 5% (v/v) CO2. At the indicated time points, 10 µL CCK-8 solution was added to each well, and then the plates were incubated for 4 h. The optical density
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(OD) was measured at 450 nm using a microplate reader (BioRad, Hercules, CA, USA). All samples were tested as five independent replicates. The index of cell
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viability in the presence of DON was calculated with the following formula: Index of viability (%) = ([OD of stimulated cells treated with DON] / [OD of stimulated cells
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without DON treatment]) × 100.
2.4 Determination of Apoptotic Cells
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Apoptosis induction by DON was analyzed by annexin V-binding and propidium iodide (PI) uptake. After treatment for 48 h, 400 µL binding buffer (BD Pharmingen) was added to the harvested cells. Resuspended cells were then incubated with 10 µl annexin V-FITC and 20 µL PI for 15 min in the dark at room temperature. Apoptosis was measured by flow cytometry on 10,000 cells per sample.
2.5 Determination of DNA Fragmentation Fragmented DNA was isolated using a DNA extraction kit (Invitrogen Biotechnology Co.) according to the manufacturer’s instructions. DNA was electrophoresed on a 1.2% (w/v) agarose gel at 100 V for 20 min. The gel was examined and photographed with an ultraviolet gel documentation system.
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2.6 ROS measurement ROS generation was monitored by measuring hydrogen peroxide (H2O2) production using the fluorescent probe, DCFH-DA. This dye is cleaved to form non-fluorescent DCFH in cells, which is oxidized to fluorescent DCF by ROS. Thus,
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the DCF fluorescence intensity is proportional to the amount of peroxide produced by
cells. Briefly, 1.5 × 106 harvested cells/mL were incubated with DCFH-DA (100 µM
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final concentration) for 30 min in the dark at 37°C. After treatment, cells were immediately washed twice and resuspended in PBS. The relative fluorescence
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intensity of ROS generation was measured using a flow cytometer on 10,000 cells per
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sample.
2.7 Detection of MMP ΔΨm
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Rhodamine 123 is a lipophilic cationic fluorescent dye that is incorporated into mitochondria in a transmembrane potential-dependent manner. The dye is selectively
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taken up by mitochondria, and its uptake is directly proportional to the MMP ΔΨm of cells (Scaduto and Grotyohann, 1999). After treatment for 48 h, harvested cells were
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incubated with rhodamine 123 (5 µg/ml final concentration) for 30 min in the dark at
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37°C and then harvested and resuspended in PBS. The relative MMP ΔΨm was measured by flow cytometry on 10,000 cells per sample.
2.8 Apoptosis-related protein assays The concentration of Bc1-2, p53, Bax, Bak-1, and Caspase-3 in supernatants
were measured using enzyme linked immuno-absorbent assays (ELISA)
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Bc1-2, p53, Bax, Bak-1, and Caspase-3 according to
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development kits for chicken
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the manufacture instructions.
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2.9 Quantification of Bax, Bak-1, Bcl-2, p53 and Caspase-3 mRNA
Total RNA was isolated from cells using Trizol reagent (Invitrogen Biotechnology
determined using
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Co.) according to the manufacturer’s instructions. RNA concentrations were GeneQuant 1300 (GE Healthcare Bio-Sciences, Piscataway, NJ,
USA). The reverse transcription reaction (40 µL) consisted of 10 µg total RNA, 1 µL
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M-MLV reverse transcriptase, 1 µL RNase inhibitor, 4 µL dNTP, 2 µL Oligo dT, 4 µL dithiothreitol and 8 µL 5× buffer. Reverse transcription was performed according
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stored at -80 °C until use.
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to the manufacturer’s instructions (Invitrogen Biotechnology Co.), and cDNA was
For primer design, we used chicken Bax, Bak-1, Bcl-2, p53 and Caspase-3 mRNA
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GenBank sequences with accession numbers XM_422067.2, NM_001030920.1, Z11961.1, X13057.1 and NM_204725.1, respectively. Chicken β-actin (GenBank
accession number L08165.1), a housekeeping gene, was used as an internal reference. Primers (Table 1) were designed using Prime 5 Software (Molecular Biology Insights, Inc., Cascade, CO, USA) and were synthesized by Invitrogen Biotechnology Co. Ltd.. Real-time PCR was performed to detect the expression of Caspase-3 and β-actin
genes using SYBR Premix Ex Taq (TaKaRa). Real-time PCR was performed using the ABI PRISM 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The PCR program consisted of 95°C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Dissociation curves were analyzed using Dissociation Curves 1.0 Software (Applied Biosystems) to detect possible primer-dimer and nonspecific amplification. Results as indicated by fold changes were expressed using
Page 9 of 25
the Pfaffl method with the following formula (Pfaffl, 2001; Cikos et al., 2007): Ratio = (Etarget)ΔCT,target(calibrator-test)/(Eref)ΔCT,ref(calibrator-test), where ΔCT,target(calibrator-test) = (CTtarget)control group – (CTtarget)treatment group, ΔCT,ref(calibrator-test) = (CTref)control group – (CTref)treatment group, Etarget is the real-time PCR efficiency of the target gene transcript,
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and Eref is the real-time PCR efficiency of the reference gene transcript.
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2.9 Statistical Analysis
Statistical analyses of all data were performed using SAS procedures (SAS
ANOVA followed by
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Institute Inc, NC). Differences in mean RT-PCR results were analyzed using one-way Tukey’s post-test. All values were expressed as mean ±
RESULTS
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3.
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standard deviation (S.D.). A P < 0.05 was considered statistically difference.
3.1 Effect of DON on Chicken Splenic Lymphocyte Proliferation
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Cellular activity, as measured by the CCK-8 bioassay, was dose-dependently
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inhibited after treating splenic lymphocytes with 0–50 µg/mL DON and 12.5 µg/mL ConA for 48 h (Figure 1). For DON-treated cells, the IC50 value, which is the
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concentration required to inhibit cell viability by 50%, was 30.82 ± 10.48 µg/mL. 3.2 DON-induced Apoptosis and Necrosis in Splenic Lymphocytes In the present study, flow cytometric analysis (annexin V-FITC and PI double
staining) was used to quantify the extent of apoptosis and necrosis in the total cell population. Significant differences were observed in the number of apoptotic and necrotic cells between control and DON-treated cells (Figure 2). After incubation with 0.2, 0.8, 3.2, 12.5 and 50.0 µg/mL DON for 48 h, the percentage of apoptotic cells increased to 28.6%, 34.3%, 40.7%, 55.2% and 57.2%, respectively, compared with 8.4% in the control group. The percentage of necrotic cells increased to 4.0%, 4.6%, 6.2%, 9.0% and 10.5%, respectively, compared with 2.3% in the control group. These results indicate that DON increased both apoptosis and necrosis in cells as compared with the control (P < 0.01, Figure 2.A and Figure 2.B), and that the proportion of
Page 10 of 25
apoptotic cells was significantly higher than that of necrotic cells. The induction of apoptosis was confirmed by DNA gel electrophoresis. As shown in Figure 2.C, the classic laddering pattern of inter-nucleosomal DNA fragmentation was observed,
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indicating that irreversible apoptotic death had been induced. 3.3 Induction of Intracellular ROS by DON
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A significant increase in DCF fluorescence intensity was observed in splenic
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lymphocytes treated with 20 µM H2O2 as a positive control (P < 0.01). When splenic lymphocytes were incubated with 0.2, 0.8, 3.2, 12.5 or 50.0 µg/mL DON for 48 h, the intracellular ROS level was significantly increased compared with that in the control
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(ConA-stimulated only, P < 0.01) as shown in Figure 3. Notably, the DCF
was 5–6-fold higher than the control.
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3.4DON Causes Loss of MMP ΔΨm
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fluorescence intensity in splenic lymphocytes at the highest dose of DON (50 µg/mL)
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As shown in Figure 4, after treatment with DON, cells exhibited much lower fluorescence intensity than controls, indicating that DON significantly decreased
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MMP ΔΨm associated with splenic lymphocyte apoptosis. Taken together with the apoptosis data, these results suggest that the mitochondrial pathway plays a central role in DON-induced apoptosis.
3.5 Concentrations of Apoptotic Gene
The concentrations of Bax, Bak-1, Bcl-2, p53 and Caspase-3 in supernatants are
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shown in Table 2. Cells treated with 0.8 µg/mL DON had significantly higher levels
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of Bax and Caspase-3 compared with the control group (P < 0.01). Treatment with 0.2
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µg/mL DON significantly increased the levels of Bak-1 compared with the control
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group (P < 0.01). The levels of p53 increased with increasing DON concentrations (P < 0.05 or P < 0.01). Whereas levels of Bcl-2 decreased with increasing DON
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concentrations (P < 0.01, except with o.8 µg/mL DON).
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3.6Relative Quantification of Apoptotic Gene Expression
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To determine whether DON-induced apoptosis is mediated through modulation of
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Bax, Bak-1, Bcl-2, p53 or Caspase-3 gene expression, we analyzed their respective mRNA levels by RT-PCR. Figure 5 shows that splenic lymphocytes expressed Bax, Bak-1, Bcl-2, p53 and Caspase-3 mRNA. Notably, the relative expression of Bak-1, p53, Caspase-3 and the ratio of Bax/Bcl-2 in splenic lymphocytes treated with more than 0.8 µg/mL DON was significantly higher than the control (P < 0.01). Whereas
the expression of Bcl-2 was significantly lower than the control (P < 0.01).
4. DISCUSSION In the present study, we were able to demonstrate that DON-triggered oxidative stress induced cytotoxicity in chicken splenic lymphocytes in vitro. This initiation of oxidative stress is one of the factors that inhibits the ability of DNA repair, induces DNA damage, and leads to inappropriate expression of apoptosis-related proteins. ROS plays an important role in the toxic injury of cells (Thannickal and Fanburg,
Page 12 of 25
2000). ROS can damage mitochondrial macromolecules either at or near the site of their formation. Therefore, in addition to the role of mitochondria as a source of ROS, mitochondria themselves can be damaged by ROS (Earnshaw et al., 1999). Loss of Δψm is common in apoptosis and is the earliest change in mitochondria-mediated
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apoptosis. Moreover, ROS induces loss of Δψm (Zhao et al., 2010). Oxidative stress is a key factor for mitochondrial shape and dynamics (Pletjushkina et al., 2006). Our
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study has shown that DON treatment increased ROS levels in chicken splenic
lymphocytes. These results are consistent with related studies that show DON
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increases ROS levels in MTEC1 cells (Li et al, 2014). In addition, we also measured the activities of SOD, CAT, GSH-Px, and GSH and MDA content in chicken
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splenic lymphocytes (data not shown). Compared with the control group, the activities of SOD, CAT, GSH-Px and GSH levels in the exposed groups decreased
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significantly (P < 0.05 or P < 0.01), but the concentration of MDA increased significantly (P
S1382-6689(14)00298-1 http://dx.doi.org/doi:10.1016/j.etap.2014.11.028 ENVTOX 2145
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
3-6-2014 11-11-2014 15-11-2014
Please cite this article as: Ren, Z., Wang, Y., Deng, H., Deng, Y., Deng, J., Zuo, Z., Wang, Y., Peng, X., Cui, H., Shen, L.,Deoxynivalenol induces apoptosis in chicken splenic lymphocytes via the reactive oxygen speciesmediated mitochondrial pathway, Environmental Toxicology and Pharmacology (2014), http://dx.doi.org/10.1016/j.etap.2014.11.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DON inhibited the growth of splenic lymphocytes in a dose-dependent manner. Cytotoxicity was assessed with the CCK-8 bioassay. DON treatment induced ROS accumulation in splenic lymphocytes. Apoptosis was correlated with inappropriate of p53, Bax, Bak-1, Bcl-2, and caspase-3 concentrations. 5. Apoptosis was correlated with disordered of p53, Bax, Bak-1, Bcl-2, and caspase-3 expression.
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1. 2. 3. 4.
Page 1 of 25
Deoxynivalenol induces apoptosis in chicken splenic lymphocytes via the reactive oxygen species-mediated mitochondrial pathway Zhihua Ren1, Yachao Wang1, 2, Huidan Deng1, Youtian Deng1, Junliang Deng1*, Zhicai Zuo1, Ya
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Wang1, Xi Peng1, Hengmin Cui1, Liuhong Shen1
1 College of Veterinary Medicine, Sichuan Agricultural University; Sichuan Province Key
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Laboratory of Animal Disease ＆ Human Health; Key Laboratory of Environmental Hazard and Human Health of Sichuan Province, Ya’an, 625014, China;
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2 School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, 621010, China.
an
Email address:
Zhihua Ren (Doctor, associate Professor): [email protected]
M
Yachao Wang (Doctor, Lecturer): [email protected] Huidan Deng (Master): [email protected]
Youtian Deng (Undergraduate):[email protected]
d
corresponding author (Doctor, Professor) : Junliang Deng: [email protected]
te
Zhicai Zuo (Doctor, Professor): [email protected] Ya Wang (Master, experimentalist): [email protected]
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Xi Peng (Doctor, Professor): [email protected] Hengmin Cui (Doctor, Professor): [email protected] Liuhong Shen (Doctor, associate Professor): [email protected]
E-mail address of corresponding author: [email protected] (J. L. Deng). Telephone: +86 835 2885313; fax:+86 835 2885302.
Postal address of corresponding author: College of Veterinary Medicine, Sichuan Agricultural University, Ya’an, Sichuan 625014, China **Z. H. Ren and Y. C. Wang contributed equally to this work and should be considered co-first authors.
The authors confirm that this manuscript has not been published elsewhere and is not under
Page 2 of 25
consideration by another journal. All authors have approved the manuscript and agree with submission to the Toxicology in Vitro. The authors have no conflicts of interest to declare.
ip t
ABSTRACT We investigated the immunotoxicity and cytotoxicity of deoxynivalenol (DON), a
mycotoxin, and the mechanism by which it induces apoptosis. Chicken splenic
cr
lymphocytes treated with 0-50 µg/mL DON for 48 h inhibited growth of splenic
us
lymphocytes in a dose-dependent manner, as revealed by the Cell Counting Kit-8 (CCK-8) bioassay. Annexin V-fluorescein isothiocyanate staining indicated that the number of apoptotic and necrotic cells were significantly higher compared with the
an
control (P < 0.01). DON treatment induced ROS accumulation, resulting in reduced mitochondrial transmembrane potential, as detected by flow cytometry and 2’,
M
7’-dichlorofluorescein acetate and rhodamine 123 labeling, respectively. Enzyme linked immunosorbent assays revealed that the concentrations of p53, Bax, Bak-1, and
d
Caspase-3 increased with increasing DON concentration (P < 0.05 or P < 0.01),
te
whereas the concentrations of Bcl-2 decreased (P < 0.01) compared with the control. These data suggest that DON induces apoptosis in splenic lymphocytes via a
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ROS-mediated mitochondrial pathway. Keywords: Deoxynivalenol; apoptosis; mitochondria; reactive oxygen species
Abbreviations:
CCK-8, Cell Counting Kit-8; ConA, concanavalin A; DON, Deoxynivalenol; ELISA, enzyme linked immunosor bent assay; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HO, hydroxyl radicals; H2O2, hydrogen peroxide; METC1, mouse
thymic epithelial cell line 1; MMP ΔΨm, mitochondrial membrane potential; O2−, superoxide; OD, optical density; PBS, phosphate-buffered saline; PI, propidium iodide; ROS, reactive oxygen species; SD, standard deviation;
Page 3 of 25
1.
INTRODUCTION Mycotoxins are secondary metabolites of molds, which contaminate a wide range
of crop plants and fruits before or after harvest. Aflatoxins, such as deoxynivalenol
ip t
(DON), ochratoxin A, fumonisin, zearalenone, patulin and T-2 toxin, are among the most common mycotoxins. They are characteristically stable under changing
cr
environmental conditions and cause various toxic effects in experimental animals, livestock and humans (Chen et al., 2008).
us
DON, also known as vomitoxin, is a mycotoxin classified as a type B trichothecene, which is produced mainly by Fusarium graminearum and F. culmorum.
an
DON is present at toxicologically relevant concentrations in cereals and grains worldwide. Furthermore, DON is resistant to milling, processing and heating, and
M
therefore readily enters both the human and animal food chains (Jackson et al., 1999; Rotter et al., 1996). To date, DON has been shown to exhibit toxic effects in humans
d
and all animal species, impairing growth in experimental animals and inducing dysregulation of the immune system (Accensi et al., 2006; Pestka et al., 2005; Pinton
te
et al., 2008). Depending on the dose and frequency of exposure, DON can be either
Ac ce p
immunosuppressive or immunostimulatory. At the cellular level, DON interacts with peptidyl transferase in the 60S ribosomal subunit and triggers ribotoxic stress (Pestka, 2008). The multiple cellular effects of trichothecenes on eukaryotic cells have been reviewed elsewhere (Rocha et al., 2005). In summary, trichothecenes inhibit protein, DNA and RNA synthesis, and mitochondrial function, and they also affect cell division, membrane integrity, and induce apoptosis. Lymphoid cells and fibroblasts are the most DON-sensitive cell types. One of the most important effects of the trichothecene mycotoxins is impairment of immune function. However, most in vivo immunotoxicity studies have been conducted in the mouse or pig (Goyarts et al, 2006; Tiemann et al, 2006). In contrast, numerous DON cytotoxicity studies have been conducted in humans, laboratory animals (mice, rats, and guinea pigs) and domestic pigs (Pestka et al., 2005; Calvert et al., 2005; Cetin and Bullerman, 2005; Fornelli et al., 2004; Minervini et al., 2004;
Page 4 of 25
Pestka, 2007). Many studies have suggested that the DON-induced production of free radicals may be one of the mechanisms that cause damage to the cellular membrane and to DNA. Thus, oxidative stress is regarded as an important factor in DON-induced toxicity (Rizzo et al., 1994). Recent studies have demonstrated that
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DON is able to decrease cell viability and cause damage to the cellular membrane,
chromosomes, or DNA. It is also able to induce lipid peroxidation and raise the levels
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of 8-OHdG and ROS in human peripheral blood lymphocytes (Yang et al., 2014). However, to our knowledge, studies on DON-induced apoptosis in chicken cells are
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limited. The present study evaluates the effects of DON in primary chicken splenic
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lymphocytes and explores the mechanism of apoptosis induction.
2. 2. MATERIALS AND METHODS 2.1 Chemicals
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DON, RPMI 1640 medium, and 2’,7’-dichlorofluorescein diacetate (DCFH-DA) and rhodamine 123 were purchased from Sigma-Aldrich (St Louis, MO, USA).
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Histopaque 1077 and the Cell Counting Kit-8 (CCK-8) were obtained from Dojindo Laboratories (Tokyo, Japan). The annexin V-fluorescein isothiocyanate (FITC)
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Apoptosis Detection kit was purchased from BD Pharmingen (Lexington, KY, USA). The chicken Bc1-2, p53, Bax, Bak-1, and Caspase-3 ELISA kits were obtained from
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Qiyi Biological Technology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) was purchased from Sijiqing Biological Engineering Materials Co., Ltd. (Hangzhou, China). Trizol reagent and the DNA extraction kit were purchased from Invitrogen Biotechnology Co., Ltd. (Shanghai, China). The SYBR PremixScript RT-PCR Kit II was purchased from Takara (Shiga, Japan). All other reagents used were of analytical grade.
2.2 Cell Isolation and Culture All procedures in this study were approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University. One hundred and eighty 1-day-old healthy male Isa Brown chickens were obtained from a comercial rearing farm (Jilin poultry farm, Jilin province) at day of hatching. One-day old chickens were placed in
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large pens with wood shavings and were reared with lighting regimen 23 h light and 1 h dark. The initial room temperature of 32-33 °C was reduced weekly by 1 °C to a final temperature of 28 °C. The relative humidity was within a range 50-60%. The chickens provided with water as well as feed ad libitum. And the chickens were fed
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only a basic commercial diet. Nutritional requirements were adequate according to
National Research Council (NRC, 1994) and the Chinese Feeding Standard of
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Chickens (NY/T33-2004). At 8 weeks of age, chickens in good health were
anaesthetized with an intramuscular injection of 846 anesthetic mixture (haloperidol,
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dihydroetorphine, and 2, 4 - dimethylaniline thiazole) (Duan et al, 2005) using doses 0.8 mL/kg body wright, respectively. After laparotomy, spleen samples of chickens
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were removed from the experimental animal.
The spleens were ground on ice and teased through a 200-mesh cell strainer into a Petri dish containing phosphate-buffered saline (PBS). The cell suspension was
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overlaid onto Histopaque 1077 and centrifuged at 400 × g for 15 min at room temperature. Lymphocytes at the interface were collected, washed twice with PBS by
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centrifugation at 250 × g for 5 min at room temperature, and then suspended in RPMI-1640 medium (without phenol red) supplemented with 10% (v/v) FBS, 100
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U/mL penicillin, and 100 U/mL streptomycin. More than 95% of cells were viable
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based on trypan blue dye exclusion.
To monitor various parameters (excluding the CCK-8 bioassay), splenic
lymphocytes were cultured in six-well culture plates (6 × 106 cells/mL) in
quintuplicate, stimulated with 12.5 µg/mL concanavalin A (ConA) to induce cell proliferation and treated with 0.0, 0.2, 0.8, 3.2, 12.5 or 50.0 µg/mL DON at 41.5 °C in
a humidified atmosphere with 5% (v/v) CO2 for 48 h (Okamura et al., 2004). After 48 h of incubation, the supernatants were collected for Bc1-2, p53, Bax, Bak-1, and Caspase-3 analysis. Cells were collected to analyze the rate of apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP ΔΨm) and for DNA and RNA isolation. Both the supernatants and the cells were frozen at
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80 °C until use.
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2.3 CCK-8 Bioassay
Cell viability was quantified using the CCK-8 bioassay as described elsewhere
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(Miyamoto et al., 2002). Splenic lymphocytes were seeded into 96-well culture plates
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(6 × 105/well) and treated with 0–50 µg/mL DON and 12.5 µg/ml ConA for 48 h at 41.5 °C with 5% (v/v) CO2. At the indicated time points, 10 µL CCK-8 solution was added to each well, and then the plates were incubated for 4 h. The optical density
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(OD) was measured at 450 nm using a microplate reader (BioRad, Hercules, CA, USA). All samples were tested as five independent replicates. The index of cell
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viability in the presence of DON was calculated with the following formula: Index of viability (%) = ([OD of stimulated cells treated with DON] / [OD of stimulated cells
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without DON treatment]) × 100.
2.4 Determination of Apoptotic Cells
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Apoptosis induction by DON was analyzed by annexin V-binding and propidium iodide (PI) uptake. After treatment for 48 h, 400 µL binding buffer (BD Pharmingen) was added to the harvested cells. Resuspended cells were then incubated with 10 µl annexin V-FITC and 20 µL PI for 15 min in the dark at room temperature. Apoptosis was measured by flow cytometry on 10,000 cells per sample.
2.5 Determination of DNA Fragmentation Fragmented DNA was isolated using a DNA extraction kit (Invitrogen Biotechnology Co.) according to the manufacturer’s instructions. DNA was electrophoresed on a 1.2% (w/v) agarose gel at 100 V for 20 min. The gel was examined and photographed with an ultraviolet gel documentation system.
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2.6 ROS measurement ROS generation was monitored by measuring hydrogen peroxide (H2O2) production using the fluorescent probe, DCFH-DA. This dye is cleaved to form non-fluorescent DCFH in cells, which is oxidized to fluorescent DCF by ROS. Thus,
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the DCF fluorescence intensity is proportional to the amount of peroxide produced by
cells. Briefly, 1.5 × 106 harvested cells/mL were incubated with DCFH-DA (100 µM
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final concentration) for 30 min in the dark at 37°C. After treatment, cells were immediately washed twice and resuspended in PBS. The relative fluorescence
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intensity of ROS generation was measured using a flow cytometer on 10,000 cells per
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sample.
2.7 Detection of MMP ΔΨm
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Rhodamine 123 is a lipophilic cationic fluorescent dye that is incorporated into mitochondria in a transmembrane potential-dependent manner. The dye is selectively
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taken up by mitochondria, and its uptake is directly proportional to the MMP ΔΨm of cells (Scaduto and Grotyohann, 1999). After treatment for 48 h, harvested cells were
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incubated with rhodamine 123 (5 µg/ml final concentration) for 30 min in the dark at
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37°C and then harvested and resuspended in PBS. The relative MMP ΔΨm was measured by flow cytometry on 10,000 cells per sample.
2.8 Apoptosis-related protein assays The concentration of Bc1-2, p53, Bax, Bak-1, and Caspase-3 in supernatants
were measured using enzyme linked immuno-absorbent assays (ELISA)
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Bc1-2, p53, Bax, Bak-1, and Caspase-3 according to
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development kits for chicken
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the manufacture instructions.
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2.9 Quantification of Bax, Bak-1, Bcl-2, p53 and Caspase-3 mRNA
Total RNA was isolated from cells using Trizol reagent (Invitrogen Biotechnology
determined using
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Co.) according to the manufacturer’s instructions. RNA concentrations were GeneQuant 1300 (GE Healthcare Bio-Sciences, Piscataway, NJ,
USA). The reverse transcription reaction (40 µL) consisted of 10 µg total RNA, 1 µL
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M-MLV reverse transcriptase, 1 µL RNase inhibitor, 4 µL dNTP, 2 µL Oligo dT, 4 µL dithiothreitol and 8 µL 5× buffer. Reverse transcription was performed according
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stored at -80 °C until use.
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to the manufacturer’s instructions (Invitrogen Biotechnology Co.), and cDNA was
For primer design, we used chicken Bax, Bak-1, Bcl-2, p53 and Caspase-3 mRNA
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GenBank sequences with accession numbers XM_422067.2, NM_001030920.1, Z11961.1, X13057.1 and NM_204725.1, respectively. Chicken β-actin (GenBank
accession number L08165.1), a housekeeping gene, was used as an internal reference. Primers (Table 1) were designed using Prime 5 Software (Molecular Biology Insights, Inc., Cascade, CO, USA) and were synthesized by Invitrogen Biotechnology Co. Ltd.. Real-time PCR was performed to detect the expression of Caspase-3 and β-actin
genes using SYBR Premix Ex Taq (TaKaRa). Real-time PCR was performed using the ABI PRISM 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The PCR program consisted of 95°C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Dissociation curves were analyzed using Dissociation Curves 1.0 Software (Applied Biosystems) to detect possible primer-dimer and nonspecific amplification. Results as indicated by fold changes were expressed using
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the Pfaffl method with the following formula (Pfaffl, 2001; Cikos et al., 2007): Ratio = (Etarget)ΔCT,target(calibrator-test)/(Eref)ΔCT,ref(calibrator-test), where ΔCT,target(calibrator-test) = (CTtarget)control group – (CTtarget)treatment group, ΔCT,ref(calibrator-test) = (CTref)control group – (CTref)treatment group, Etarget is the real-time PCR efficiency of the target gene transcript,
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and Eref is the real-time PCR efficiency of the reference gene transcript.
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2.9 Statistical Analysis
Statistical analyses of all data were performed using SAS procedures (SAS
ANOVA followed by
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Institute Inc, NC). Differences in mean RT-PCR results were analyzed using one-way Tukey’s post-test. All values were expressed as mean ±
RESULTS
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3.
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standard deviation (S.D.). A P < 0.05 was considered statistically difference.
3.1 Effect of DON on Chicken Splenic Lymphocyte Proliferation
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Cellular activity, as measured by the CCK-8 bioassay, was dose-dependently
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inhibited after treating splenic lymphocytes with 0–50 µg/mL DON and 12.5 µg/mL ConA for 48 h (Figure 1). For DON-treated cells, the IC50 value, which is the
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concentration required to inhibit cell viability by 50%, was 30.82 ± 10.48 µg/mL. 3.2 DON-induced Apoptosis and Necrosis in Splenic Lymphocytes In the present study, flow cytometric analysis (annexin V-FITC and PI double
staining) was used to quantify the extent of apoptosis and necrosis in the total cell population. Significant differences were observed in the number of apoptotic and necrotic cells between control and DON-treated cells (Figure 2). After incubation with 0.2, 0.8, 3.2, 12.5 and 50.0 µg/mL DON for 48 h, the percentage of apoptotic cells increased to 28.6%, 34.3%, 40.7%, 55.2% and 57.2%, respectively, compared with 8.4% in the control group. The percentage of necrotic cells increased to 4.0%, 4.6%, 6.2%, 9.0% and 10.5%, respectively, compared with 2.3% in the control group. These results indicate that DON increased both apoptosis and necrosis in cells as compared with the control (P < 0.01, Figure 2.A and Figure 2.B), and that the proportion of
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apoptotic cells was significantly higher than that of necrotic cells. The induction of apoptosis was confirmed by DNA gel electrophoresis. As shown in Figure 2.C, the classic laddering pattern of inter-nucleosomal DNA fragmentation was observed,
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indicating that irreversible apoptotic death had been induced. 3.3 Induction of Intracellular ROS by DON
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A significant increase in DCF fluorescence intensity was observed in splenic
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lymphocytes treated with 20 µM H2O2 as a positive control (P < 0.01). When splenic lymphocytes were incubated with 0.2, 0.8, 3.2, 12.5 or 50.0 µg/mL DON for 48 h, the intracellular ROS level was significantly increased compared with that in the control
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(ConA-stimulated only, P < 0.01) as shown in Figure 3. Notably, the DCF
was 5–6-fold higher than the control.
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3.4DON Causes Loss of MMP ΔΨm
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fluorescence intensity in splenic lymphocytes at the highest dose of DON (50 µg/mL)
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As shown in Figure 4, after treatment with DON, cells exhibited much lower fluorescence intensity than controls, indicating that DON significantly decreased
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MMP ΔΨm associated with splenic lymphocyte apoptosis. Taken together with the apoptosis data, these results suggest that the mitochondrial pathway plays a central role in DON-induced apoptosis.
3.5 Concentrations of Apoptotic Gene
The concentrations of Bax, Bak-1, Bcl-2, p53 and Caspase-3 in supernatants are
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shown in Table 2. Cells treated with 0.8 µg/mL DON had significantly higher levels
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of Bax and Caspase-3 compared with the control group (P < 0.01). Treatment with 0.2
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µg/mL DON significantly increased the levels of Bak-1 compared with the control
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group (P < 0.01). The levels of p53 increased with increasing DON concentrations (P < 0.05 or P < 0.01). Whereas levels of Bcl-2 decreased with increasing DON
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concentrations (P < 0.01, except with o.8 µg/mL DON).
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3.6Relative Quantification of Apoptotic Gene Expression
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To determine whether DON-induced apoptosis is mediated through modulation of
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Bax, Bak-1, Bcl-2, p53 or Caspase-3 gene expression, we analyzed their respective mRNA levels by RT-PCR. Figure 5 shows that splenic lymphocytes expressed Bax, Bak-1, Bcl-2, p53 and Caspase-3 mRNA. Notably, the relative expression of Bak-1, p53, Caspase-3 and the ratio of Bax/Bcl-2 in splenic lymphocytes treated with more than 0.8 µg/mL DON was significantly higher than the control (P < 0.01). Whereas
the expression of Bcl-2 was significantly lower than the control (P < 0.01).
4. DISCUSSION In the present study, we were able to demonstrate that DON-triggered oxidative stress induced cytotoxicity in chicken splenic lymphocytes in vitro. This initiation of oxidative stress is one of the factors that inhibits the ability of DNA repair, induces DNA damage, and leads to inappropriate expression of apoptosis-related proteins. ROS plays an important role in the toxic injury of cells (Thannickal and Fanburg,
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2000). ROS can damage mitochondrial macromolecules either at or near the site of their formation. Therefore, in addition to the role of mitochondria as a source of ROS, mitochondria themselves can be damaged by ROS (Earnshaw et al., 1999). Loss of Δψm is common in apoptosis and is the earliest change in mitochondria-mediated
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apoptosis. Moreover, ROS induces loss of Δψm (Zhao et al., 2010). Oxidative stress is a key factor for mitochondrial shape and dynamics (Pletjushkina et al., 2006). Our
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study has shown that DON treatment increased ROS levels in chicken splenic
lymphocytes. These results are consistent with related studies that show DON
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increases ROS levels in MTEC1 cells (Li et al, 2014). In addition, we also measured the activities of SOD, CAT, GSH-Px, and GSH and MDA content in chicken
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splenic lymphocytes (data not shown). Compared with the control group, the activities of SOD, CAT, GSH-Px and GSH levels in the exposed groups decreased
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significantly (P < 0.05 or P < 0.01), but the concentration of MDA increased significantly (P
