Toxicology 333 (2015) 76–88

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Gene expression profiling identifies the novel role of immunoproteasome in doxorubicin-induced cardiotoxicity Wen-Jie Zhao a,b,c , Sheng-Nan Wei a,b,c, Xiang-Jun Zeng a, ** , Yun-Long Xia d , Jie Du a,b,c , Hui-Hua Li a,b,c, * a Department of Physiology and Pathophysiology, Beijing Anzhen Hospital the Key Laboratory of Remodeling-Related Cardiovascular Diseases, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China b Department of Cardiology, Chaoyang Hospital, Capital Medical University, Beijing 100069, China c Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing 100029, China d Department of Cardiology, Institute of Cardiovascular Diseases, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 February 2015 Received in revised form 13 April 2015 Accepted 14 April 2015 Available online 17 April 2015

The most well-known cause of chemotherapy-induced cardiotoxicity is doxorubicin (DOX). The ubiquitin-proteasome system (UPS) is the main cellular machinery for protein degradation in eukaryotic cells. However, the expression pattern of the UPS in DOX-induced cardiotoxicity remains unclear. C57BL/ 6 mice were intraperitoneally injected with a single dose of DOX (15 mg/kg). After 1, 3 and 5 days, cardiac function and apoptosis were detected with echocardiography and TUNEL assay. Microarray assay and qPCR analysis were also performed at day 5. We found that DOX caused a significant decrease in cardiac function at day 5 and increase in cardiomyocyte apoptosis at days 3 and 5. Microarray data revealed that totally 1185 genes were significantly regulated in DOX-treated heart, and genes involved in apoptosis and the UPS were mostly altered. Among them, the expression of 3 immunoproteasome catalytic subunits (b1i, b2i and b5i) was markedly down-regulated. Moreover, DOX significantly decreased proteasome activities and enhanced polyubiquitinated proteins in the heart. Importantly, overexpression of immunoproteasome catalytic subunits (b1i, b2i or b5i) significantly attenuated DOX-induced cardiomyocyte apoptosis and other UPS gene expression while knockdown of them significantly increased DOX-induced cardiomyocyte apoptosis. These effects were partially associated with increased degradation of multiple pro-apoptotic proteins. In conclusion, our studies suggest that immunoproteasome plays an important role in DOX-induced cardiomyocyte apoptosis, and may be a novel therapeutic target for prevention of DOX-induced cardiotoxicity. ã 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cardiotoxicity Doxorubicin Apoptosis Ubiquitin-proteasome system Immunoproteasome Microarray

1. Introduction DOX is one of the most effective anticancer agents of a variety of solid tumors but its use is limited primarily due to cardiotoxicity

Abbreviations: DOX, doxorubicin; ROS, reactive oxygen species; UPS, ubiquitinproteasome system; E1s, ubiquitin activating enzymes; E2s, ubiquitin conjugating enzymes; E3s, ubiquitin ligases; DUBs, deubiquitinases. * Corresponding author at: Department of Physiology and Pathophysiology, Capital Medical University, No 10 Xitoutiao, You An Men, Beijing 100069, China. Tel.: +861083950091; fax:+861083950091 ** Corresponding author at: Department of Physiology and Pathophysiology, Beijing Anzhen Hospital the Key Laboratory of Remodeling-Related Cardiovascular Diseases, Department of Cardiology, Beijing Chaoyang Hospital, Capital Medical University, No.10 Xitoutiao, You An Men, Beijing 100069, China. Tel.:+861083950090; fax: +86 10 83950091. E-mail addresses: [email protected] (X.-J. Zeng), [email protected] (H.-H. Li). http://dx.doi.org/10.1016/j.tox.2015.04.009 0300-483X/ ã 2015 Elsevier Ireland Ltd. All rights reserved.

(Carvalho et al., 2014; Chatterjee et al., 2010; Octavia et al., 2012; Ranek and Wang, 2009). DOX-induced cardiotoxicity may be acute or chronic. The incidence of acute cardiotoxicity is much higher than chronic cardiotoxicity, which are approximately 11% and 1.7%, respectively. They develop dilated cardiomyopathy and congestive heart failure eventually (Carvalho et al., 2014; Chatterjee et al., 2010). Emerging evidence demonstrates multiple mechanisms for cardiotoxic effects of DOX which include the generation of ROS, membrane lipid peroxidation, mitochondrial dysfunction, decreased activity of Na+–K+ ATPase, intracellular calcium dysregulation and these cellular changes eventually lead to myocardial apoptosis and cardiac dysfunction (Carvalho et al., 2014; Chatterjee et al., 2010; Octavia et al., 2012). Recently, increased UPS activity has been reported to be associated with DOX-induced cardiotoxicity (Ranek and Wang, 2009; Shi et al., 2011). While Sishi et al. demonstrated that DOX decreased chymotrypsin-like activity and increased protein ubiquitination in the heart (Sishi et al., 2013).

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The UPS plays a key role in mediating the degradation of intracellular proteins (Wang and Robbins, 2014). UPS-mediated proteolysis includes 2 major steps: ubiquitination and proteasome-mediated degradation. Ubiquitination is a series of enzymatic reactions involving the E1s, E2s and E3s (Powell et al., 2012; Wang and Robbins, 2014). DUBs hydrolyze ubiquitin chains to make ubiquitin molecular to proceed to next ubiquitination process (Wang and Robbins, 2014). Finally, the polyubiquitinated proteins are rapidly degraded by the 26S proteasome. Dysfunction of the UPS has been implicated in numerous cardiovascular diseases, such as atherosclerosis, myocardial ischemia, hypertrophy and heart failure (Li and Wang, 2011; Powell et al., 2012). And it has been revealed that UPS is correlated with cardiomyocyte apoptosis by degradation of pro-survival transcriptional factors (Ranek and Wang, 2009). The 26S proteasome consists of a 20S core particle and two regulatory particles (Tian et al., 2012). The 20S core harbors the three constitutive catalytic subunits b1, b2 and b5 in the inner b-rings (Angeles et al., 2012; Tian et al., 2012). However, in response to inflammatory cytokines such as interferon-g (IFN-g), the constitutively expressed catalytic b-subunits (b1, b2 and b5) are replaced by inducible b-counterparts known as immunosubunits (Angeles et al., 2012; Ferrington and Gregerson, 2012). These immunosubunits, including b1i (also known as LMP2 or PSMB9), b2i (also known as MECL-1 or PSMB10) and b5i (also known as LMP7 or PSMB8), are preferentially incorporated during proteasome assemble to form the immunoproteasome (Angeles et al., 2012; Ferrington and Gregerson, 2012). Now it is known that immunoproteasome-mediated proteolysis has emerged as a critical molecular mechanism for regulating both the innate and adaptive immune responses, protecting against oxidative stress and maintaining cellular protein homeostasis (Angeles et al., 2012). But it is still unclear which components of UPS play a key role in DOX-induced cardiotoxicity. Therefore, the present study aimed to search for the altered components of the UPS and to determine the role of immunoproteasome expression in DOX-induced cardiotoxocity. 2. Materials and methods 2.1. Antibodies and reagents Anti-ubiquitin antibody was purchased from Millipore (Temecula, CA, USA). Anti-b1, b5, a-actinin and b-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-b2, b1i, b5i and PA28a antibodies were from Abcam (Cambridge, UK). Anti-b2i antibody was purchased from Enzo Life Science (Farmingdale, NY, USA). Anti-Bcl-2 and Bax Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Apoptosis detection kit (DeadEndTM Fluorometric TUNEL System) was purchased from Promega (Madison, WI, USA). Fluorescent substrates Suc-LLVY-AMC, Z-LLE-AMC and Ac-RLR-AMC, proteasome inhibitor epoxomicin and MG132 were purchased from Boston Biochem (Cambridge, MA, USA). Ac-DEVD-AFC was acquired from Biomol (Plymouth Meeting, PA, USA). The DMEM/F12 culture medium and fetal bovine serum were obtained from Hyclone (Thermo; USA). Doxorubicin and other reagents were purchased from Sigma– Aldrich (Louis, MO, USA). 2.2. Animals and treatment Male 8-week-old C57BL/6 mice were administered intraperitoneally with a single dose of 15 mg/kg body weight of DOX. In control mice, the same volume of saline was injected (Li et al.,

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2006; Zhu et al., 2011). All mice underwent echocardiography measurements at days 1, 3 and 5. Then the mice were anesthetized by an overdose of pentobarbital (100 mg/kg, i.p.) and heart tissues were quickly harvested and prepared for further histological and molecular analysis. This study was approved by the Institutional Animal Care and Use Committee of Capital Medical University and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. 2.3. Cardiac echocardiography detection The hair of left chest wall was removed by depilatory cream. The mouse was placed in the mouse anesthesia induction chamber to induce anesthesia (the oxygen level: 1 L/min; the concentration of isoflurane: 2.5%). And then masked and reset the concentration of isoflurane to 1–1.5% to maintain the anesthesia. The mouse paws were placed on the ECG electrode contact pads with dermatological tape. Echocardiograms were obtained by transthoracic echocardiograpy using metal ECG connector. We measured the left ventricular anterior wall thickness (LVAW), the left ventricular posterior wall thickness (LVPW) and the left ventricular internal diameter in systole (s) and diastole (d), respectively. And then we calculated ejection fraction (EF) and fractional shortening (FS) as the following formula: FS% = (LVID;d-LVID;s)/LVID;d
100%; LV Vol;d = [7.0/(2.4 + LVID;d)]
LVID;d3
1000; LV Vol;s = [7.0/ (2.4 + LVID;s)]
LVID;s3
1000; EF% = (LV Vol;d-LV Vol;s)/LV vol; d
100%. 2.4. Primary cardiomyocyte culture and adenovirus infection Recombinant adenovirus expressing GFP alone (Ad-GFP), b1i and GFP (Ad-b1i), b2i and GFP (Ad-b2i), b5i and GFP (Ad-b5i) were constructed using the AdMax adenoviral expression system by Genechem. The RNA interference negative control adenovirus (Ad-shC) expressed scrambled shRNA: TTCTCCGAACGTGTCACGT. The RNA interference shRNA sequence of b1i, b2i, b5i was respectively ACCATCATGGCTGTGGAAT (Ad-shb1i), GGCTTCTCTTTCGAGAACT (Ad-shb2i) and GGAATGCAGGCTATACTAT (Ad-shb5i). Recombinant adenovirus expressing shRNA was generated using the AdMax adenoviral expression system. Neonatal rat cardiomyocytes (NRCMs) were isolated by enzymatic disassociating hearts of 1–3-day-old Sprague-Dawley (SD) rats as described (Liu et al., 2014). NRCMs were infected with overexpression adenovirus or RNA interference adenovirus 24 h and then stimulated by DOX (0.5, 1, 5 mM) for additional 24 h (Zhang et al., 2011). NRCMs were subjected to TUNEL assay and lysed by western blot analysis after DOX treatment. 2.5. TUNEL assay TUNEL assay was performed with the DeadEndTM Fluorometric TUNEL System according to manufacturer’s instructions. The samples were counterstained with DAPI as described (Liu et al., 2014; Wang et al., 2013). The apoptotic nuclei and the total nuclei were counted at a magnification of 100
(Olympus BX-63, Japan). The number of TUNEL-positive nuclei was calculated using Image J software. 2.6. Caspase-3 activity assay Caspase-3 activity was measured as described (Wang et al., 2013). Briefly, cells were homogenized in ice-cold lysis buffer. Samples were then incubated with assay buffer containing Ac-DEVD-AFC (50 mM) at 37  C for 1.5 h. Activity of caspase-3 was determined using a PerkinElmer 2030 Multilabel Microplate Reader at 405 nm.

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2.7. Microarray assay and analysis

2.10. Western blot analysis

Total RNA was extracted with Trizol reagent (Invitrogen) from the hearts (n = 3 per group) at day 5 after DOX injection according to manufacturer’s instructions. The quantity and purity of all RNA samples were determined by using the Nanodrop ND-1000 spectrophotometer (Thermo Fisher, Waltham, MA, US). 5 mg of total RNA was amplified with the GeneChip One-Cycle cDNA Synthesis Kit (Affymetrix) and GeneChip IVT Labeling Kit (Affymetrix) according to manufacturer’s instructions. 15 mg biotin-labeled complementary RNA were hybridized to Affymetrix GeneChip mouse Genome 430 2.0 array for 16 h, and then the gene chips were washed and stained on a Fluidics Station 450 and scanned in Affymetrix GeneChip Confocal Scanner 3000 (Zeng et al., 2013). A comprehensive bioinformatics analysis was used to enrich the dataset for genes. On the GeneChip mouse Genome 430 2.0 array, 45,000 probe sets analyze the expression of over 39,000 transcripts and variants from over 34,000 well characterized mouse genes. The gene expression data are available at the Gene Expression Omnibus (GEO) website: http://www.ncbi.nlm.nih.gov/geo/ (accession number GSE59672).

Myocardial tissues were homogenized with RIPA lysis buffer containing 10 mM PMSF and ultrasonic assisted extraction was performed on ice. Protein concentration was determined with BCA Protein Assay Kit (Thermo) (Wang et al., 2013). Then 50 mg proteins from tissue or cultured cardiomyocytes were electrophoresed by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes and blocked in 5% milk. Blots were incubated with primary antibodies as indicated at 4  C overnight and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. Blots were developed using a chemiluminescent system (Beijing Sage Creation) and signal intensities were analyzed by Image J software (Wang et al., 2013).

2.8. GO analysis The differential expression gene main function was analyzed by GO analysis as described previously (Zeng et al., 2013). For each transcript, fold change, statistical significance of differential expression and false discovery rate were calculated. To search for enrichment of specific biological processes, the genes showing significantly differential expression between the two groups were classified into functional groups. 2.9. Quantitative real-time PCR analysis (qPCR analysis) The first-strand cDNA was generated from 1–2 mg of total RNA by RT Enzyme Mix according to the manufacturer’s instructions on a PCR thermocycler (S1000 Thermal Cycler, Bio-Rad. USA) (Liu et al., 2014; Wang et al., 2013). The expression level of each gene was detected by qPCR and then normalized to GAPDH in the same sample. The primer sequences were described in Table 1.

2.11. Proteasome activity assay Samples were homogenized at 4  C in 10 volumes HEPES buffer (50 mM, pH 7.5) containing: KCl 20 mM, MgCl2 5 mM, DTT 1 mM. Cell debris was removed by centrifugation for 15 minutes at 12,000 g at 4  C (Tian et al., 2012), and the supernatants were immediately used for protein concentration assay and then determination of peptidase activities. The following synthetic fluorescent substrates: Z-LLE-AMC (45 mM), Ac-RLR-AMC (40 mM), Suc-LLVY-AMC (18 mM) were used respectively for measuring caspase-like, trypsin-like and chymotrypsin-like activities. MG-132 (20 mM) was used for inhibiting chymotrypsin-like and caspase-like activities and epoxomycin (5 mM) was used for inhibiting trypsin-like activity. The portion of peptidase activities inhibited by the proteasome inhibitor is attributed to the proteasome. A 10 mg of crude protein extract is added to 200 ml of the HEPES buffer containing the fluorescent substrates and incubated at 37  C for 1 h in 96-well white plates. The fluorescence intensity was measured using a PerkinElmer 2030 Multilabel Microplate Reader with the excitation wave length of 380 nm and emission wave length at 460 nm. 2.12. Statistical analysis Results are presented as means  SE. The number of experiments in every case is shown in figure legends. Statistical

Table 1 Primers used for quantitative real-time RT-PCR analysis. Gene

Forward primer

Reverse primer

Uba1 Ube2r2 Ube3a Hecw2 Mylip Nedd4l Herc1 Rnf144b Fbxl3 Fbxo31 Fbxo32 Cblb Crbn Tbl1xr1 Usp47 b1 b2 b5 b1i b2i b5i PA28a

50 -GCATCGTCAGTCAGGTCAAAGT-30 50 -CGGAGAAGGATGGAGTGAAG-30 50 -TGATTAGGGAGTTCTGGGAAAT-30 50 -TCCCACTGACACAAGACTCAAT-30 50 -ACTTGGCGTCTCTGTTTCTGA-30 50 -AGGCTGTGGATTGAGTTTGAAT-30 50 -ACTGAAATGGCTTGAACATCTG-30 50 -GCAGAACCTGGACAATGACA-30 50 -CAAACACTCACCGAAAGTCAAC-30 50 -GGAGTATGGCGTTTGTGAGAAC-30 50 -GCTGGATTGGAAGAAGATGTAT-30 50 -CCTTCTCCCAAGCATAAAGTGT-30 50 -GATTTTGCCAGAGTGTGTGTTG-30 50 -CCTAAGTGCTGGCGTAGATAAGA-30 50 -GTTTTGCTGCCTGAACAATCTC-30 50 - GCAGTTCACTGCCAATGCTCTC-30 50 - TGCCTTATGTCACCATGGGTTC-30 50 - CAGATCTGCTGGACTTGGGT-30 50 -GAAGAAGTCCACACCGGGACAA-30 50 - GAACGGACCTCAGCTCTACG-30 50 - GCCTATGGGGTGATGGACAG-30 50 -AGGAGGAGCGGAAGAAGC-30

50 -CATCACAAAGTCAGGCTCTACC5-30 50 -TCGTCGTCATACAAGTCGTCAT-30 50 -GCTCTGTCTGTGCCTGTTGTAA-30 50 -CCTCCTCCTCCAAAGAAGATG-30 50 -CCTCTTTGGATGTTCTCTTGATG-30 50 -TGTAGTTGTCCGTGGCAGAGTA-30 50 -CGGTAACTCCCTCTCTTGTAGC-30 50 -ACCAAGGCAATGACACCTAATC-30 50 -TCCCAAAGTAAAGATGCGTAGC-30 50 -TGTCTGTATCGGTGAAGCAGTT-30 50 -AGGAGAGAATGTGGCAGTGTTT-30 50 -CGTCATCATCTTCTACCGTGTC-30 50 -CCAGGAGATGGGTTTTGAAG-30 50 -GGCTGAATGGAAAGGAAACTG-30 50 -TCGTCCACATTACCAGAATCAC-30 50 - TGGTCTCCCAAAAGCACCTG-30 50 - AGCTGCAATAGCCTCACTCAC-30 50 - TGGAGAAACTTGAAGGCCAGG-30 50 - GACACCCGGGAATCAGAGC-30 50 - CCAGAGCCAAGGGCAGTAAA-30 50 -CCTTCATGTGGTACATGTTGACG-30 50 -AACCAGGTAGTGACCAGATTGA-30

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differences between groups were analyzed by the non-parametric tests (Kruskal–Wallis or Mann–Whitney U test) or by the parametric test one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test. Results are considered significant at p < 0.05. 3. Results 3.1. DOX causes cardiac dysfunction in mice To establish the model of acute cardiac injury, mice were received a single intraperitoneal injection of DOX (15 mg/kg). Cardiac function was measured by echocardiography at different time points. The left ventricular anterior wall (LVAW; s, LVAW; d) and left ventricular posterior wall in systole or diastole (LVPW; s, LVPW; d) were markedly reduced at days 3 and 5 (Supplementary Table 1). Moreover, DOX caused a significant cardiac dysfunction reflected by decreased EF% (25%) and FS% (30%) at day 5 compared with control. No significant decrease in cardiac function was observed at days 1 and 3 (Fig. 1 and Supplementary Table 1). 3.2. DOX increases cardiomyocyte apoptosis in the heart To determine whether DOX induces cardiomyocyte apoptosis, a series of TUNEL assays were performed on the heart sections at different time points. As shown in Fig. 2A, DOX significantly increased the number of TUNEL-positive nuclei at days 3 and 5 after DOX injection. Moreover, DOX significantly increased Bax/Bcl-2 ratio and activated caspase-3 and PARP in a timedependent manner (Fig. 2B and C). Therefore our results demonstrated that DOX (15 mg/kg) successfully induced apoptosis in mice which was consisted with published articles (Liu et al., 2004; Shizukuda et al., 2005). 3.3. DOX affects transcriptional expression profiles in the heart To elucidate the molecular mechanism of DOX-induced cardiotoxicity, we detected the gene expression profiles in

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DOX-treated heart by microarray assay. We found that total 1185 genes were differentially expressed in DOX-treated heart compared with the control heart. 747 genes were markedly up-regulated and 438 genes were significantly down-regulated after 5 days of DOX injection. Among them, 39 genes (3.3%) are involved in the ubiquitin-proteasome system (UPS) (Table 2). 62 genes (5.2%) are involved in apoptosis. 7 genes (0.6%) are involved in DNA repair. 17 genes (1.4%) are involved in cell cycle. 6 genes (0.5%) are involved in lysosome. 13 genes (1.1%) involved in protein folding and modification process (Supplementary Tables 2–7). These data suggest that the genes related to the UPS and apoptosis may play a central role in DOX-induced cardiotoxicity. To verify the reliability of microarray assay, qPCR analysis was performed for ten genes selected, including Uba1, Ube2r2, Herc1, PSMB9, Agtpbp1, Top2a, Igf1, Bcl2l1, Foxo3 and Ctsl, which were significantly different in the microarray data. Scatter plot analysis demonstrated a good correlation between microarray and qPCR data (Supplementary Fig. 1). 3.4. Effects of DOX on the expression of the UPS genes Since the UPS is the major pathway to control protein turnover, which plays a critical role in the regulating of DOX-induced cardiomyocyte apoptosis (Dimitrakis et al., 2012; Ranek and Wang 2009), we focus on how DOX affects UPS genes in the heart. We found that 39 UPS genes, including 1 E1 (Uba1), 3 E2s (Ube2b, Ube2r2, and Ube2i), 21 E3s (Rnf39, Zbtb16, Cblb, Fbxo31, Pcgf5, Crbn, Fbxo32, Nedd4l, Klhl2, Asb11, Hecw2, Enc1, Klhl24, Herc1, Mylip, Rnf144b, Rnf113a2, Fbxl3, Ube3a, Tbl1xr1, and Klhl7) and 2 DUBs (Ufdl1 and Usp47) were significantly up-regulated, whereas 8 E3s (Mkrn1, Trmt5, Asb10, Trim21, Asb2, Klhl30, Trim7, and Tnfaip8), 3 immunoproteasome catalytic subunits (b1i, b2i and b5i) and 1 proteasome activator (PA28a) were markedly down-regulated in DOX-treated heart compared with control (Table 2). To verify the expression of the UPS genes in DOX-treated heart, qPCR analysis was performed. Consistent with microarray results, DOX markedly increased the mRNA expression of Uba1, Ube2r2, Ube3a, Hecw2, Mylip, Nedd4l, Herc1, Rnf144b, Fbxl3, Fbxo31, Cblb,

Fig. 1. Echocardiographic assessment of cardiac functions. Mice were randomly divided into four groups and treated as described under Animals and treatment: control (saline), DOX 1d (15 mg/kg, for 1 day), DOX 3d (15 mg/kg, for 3 days), and DOX 5d (15 mg/kg, for 5 days). Cardiac function was measured by echocardiography. (A) Representative M-mode echocardiography. (B) Quantification of ejection fraction (EF %) and fractional shortening (FS %) in groups. All data are expressed as means  SE (control n = 9, DOX 1d n = 6, DOX 3d n = 8, DOX 5d n = 9). *p < 0.05 versus control.

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Fig. 2. Effects of DOX on apoptosis in hearts: mice were treated as Fig. 1. (A) Representative images of apoptotic cardiomyocytes detected by TUNEL assay (left, green). Nuclei were counterstained with DAPI (blue) and cardiomyocytes were stained with anti-a-actinin (red). Bar shows 50 mm. Column graph showed the quantitative analysis of TUNEL-positive nuclei. (B) The expression of Bcl-2 and Bax protein was performed after DOX injection by western blot analysis. Equal protein loading was confirmed by b-actin (left). Column graph showed the quantitative analysis of the ratio of Bax/Bcl-2 (right). (C) The activation of caspase-3 and PARP was detected by western blot analysis after DOX injection. Equal protein loading was confirmed by b-actin (left). Column graph showed the quantitative analysis of the expression of cleaved-caspase-3 and cleaved-PARP (right). All data are expressed as means  SE (n = 3 per group). *p < 0.05 versus control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Crbn, Tbl1xr1 and Usp47 (Fig. 3A), but markedly down-regulated immunoproteasome catalytic subunits (b1i, b2i and b5i) and proteasome activator (PA28a) in vivo and NRCMs (Figs. 3C and 7B). DOX did not affect standard proteasome catalytic subunits (b1, b2 and b5) (Fig. 3B). Western blot analysis further confirmed that DOX had no effect on standard proteasome catalytic subunits in vivo and NRCMs (Figs. 4A and 7D ), but markedly decreased the expression of immunoproteasome catalytic subunits (b1i, b2i and b5i) and proteasome activator (PA28a) in vivo and NRCMs (Figs. 4B and 7C). Consequently, these results indicate that there was a good

agreement between the microarray data and qPCR analysis, which demonstrated that UPS pathway was one of the targets of DOX in the heart. 3.5. DOX reduces proteasome activities and increases accumulation of polyubiquitinated proteins To determine whether decreased protein levels of immunoproteasome subunits alters proteasome activities, we performed proteasome activity assay using three fluorescent-labeled

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Table 2 The gene expression of ubiquitin-proteasome system. Gene symbol

Gene title

E1 Uba1

Ubiquitin-like modifier activating enzyme 1

1.44

4.4E-03

E2 Ube2b Ube2r2 Ube2i

Ubiquitin-conjugating enzyme E2B, Ubiquitin-conjugating enzyme E2R 2 Ubiquitin-conjugating enzyme E2I

1.71 1.45 1.45

2.4E-04 1.8E-02 2.4E-04

E3 Rnf39 Zbtb16 Cblb Fbxo31 Pcgf5 Crbn Fbxo32 Nedd4l Klhl2 Asb11 Hecw2 Enc1 Klhl24 Herc1 Mylip Rnf144b Rnf113a2 Fbxl3 Ube3a Tbl1xr1 Klhl7 Mkrn1 Trmt5 Asb10 Trim21 Asb2 Klhl30 Trim7 Tnfaip8

Ring finger protein 39 (Rnf39), mRNA Zinc finger and BTB domain containing 16 Casitas B-lineage lymphoma b F-box protein 31 Polycomb group ring finger 5 Cereblon F-box protein 32 Neural precursor cell expressed, developmentally down-regulated gene 4-like Kelch-like 2, Mayven (Drosophila) Ankyrin repeat and SOCS box-containing 11 HECT, C2 and WW domain containing E3 ubiquitin protein ligase 2 Ectodermal-neural cortex 1 Kelch-like 24 (Drosophila) Hect carboxyl terminus) domain and RCC1 (CHC1)-like domain (RLD) 1 Myosin regulatory light chain interacting protein Ring finger protein 144B Ring finger protein 113A2 F-box and leucine-rich repeat protein 3 Ubiquitin protein ligase E3A Transducin (beta)-like 1X-linked receptor 1 Kelch-like 7 (Drosophila) Makorin, ring finger protein, 1 TRM5 tRNA methyltransferase 5 homolog Ankyrin repeat and SOCS box-containing 10 Tripartite motif-containing 21 Ankyrin repeat and SOCS box-containing 2 Kelch-like 30 (Drosophila) Tripartite motif-containing 7 Tumor necrosis factor, alpha-induced protein 8

3.48 3.23 2.57 2.07 1.99 1.97 1.96 1.92 1.90 1.89 1.81 1.79 1.66 1.60 1.58 1.57 1.51 1.50 1.46 1.43 1.41 1.39 1.39 1.47 1.52 1.59 1.96 1.96 2.13

5.0E-06 1.2E-03 2.3E-03 2.0E-05 4.3E-03 2.0E-04 2.0E-04 1.6E-03 2.2E-03 1.0E-04 1.2E-03 4.0E-04 1.4E-03 3.2E-02 1.8E-03 1.6E-03 6.1E-03 2.0E-04 3.2E-03 2.0E-04 3.9E-02 3.1E-02 2.3E-02 8.7E-03 4.9E-03 2.1E-02 2.0E-04 3.5E-03 1.1E-05

Deubiquitinase Ufd1l Usp47

Ubiquitin fusion degradation 1 like Ubiquitin specific peptidase 47

1.48 1.37

9.5E-03 2.9E-03

Proteasome b1i b2i b5i PA28a

Proteasome Proteasome Proteasome Proteasome

1.61 1.89 1.64 1.69

4.0E-04 4.0E-04 4.0E-04 1.9E-03

Fold change

subunit, beta type 9 (large multifunctional peptidase 2) subunit, beta type 10 subunit, beta type 8 (large multifunctional peptidase 7) 28 subunit, alpha

peptides. We found that DOX also markedly decreased caspaselike, trypsin-like and chymotrypsin-like activities compared with control group in vivo and NRCMs (Figs. 5A and 7A ). Next, we investigated the effect of DOX on the polyubiquitinated proteins by Western blot analysis. As shown in Fig. 5B, DOX significantly increased the level of polyubiquitinated proteins compared with control, indicating that polyubiquitinated proteins cannot be degraded by proteasomes which means DOX impairs the proteasome activities in the heart. 3.6. DOX triggers cardiomyocyte apoptosis in cultured NRCMs To further confirm the effects of DOX on apoptosis and proteasome function in vitro, we treated NRCMs with different doses of DOX (0.5, 1 and 5 mM) for 24 h. TUNEL assay revealed that the number of TUNEL-positive cardiomyocyte nuclei was markedly increased in a dose-dependent manner after DOX treatment (Fig. 6A ). Moreover, we detected the expression of pro- and antiapoptotic proteins Bax and Bcl-2. Western blot analysis showed

p-value

that Bax/Bcl-2 ratio was significantly increased in DOX-treated NRCMs compared with the control (Fig. 6B). We also detected the activation of caspase-3 and the cleavage of PARP. The results showed that cleaved-caspase-3 and cleaved-PARP increased in a dose-dependent manner (Fig. 6C). 3.7. Overexpression of immunoproteasome catalytic subunits reduces DOX-induced cardiomyocyte apoptosis To determine whether overexpression of immunoproteasome catalytic subunits protects against DOX-induced apoptosis in vitro, NRCMs were infected with Ad-b1i, b2i, b5i-GFP or Ad-GFP for 24 h and subsequently treated with DOX for additional 24 h. TUNEL assay showed that cardiomyocyte apoptosis was increased to approximately 18% by stimulation with DOX, whereas overexpression of b1i, b2i or b5i markedly attenuated this effect (Fig. 8A and B). In addition, the change pattern of caspase-3 activity was consistent with apoptosis (Fig. 8C). Overexpression of b1i, b2i or b5i decreased the activation of caspase-3 and the cleavage of

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Fig. 3. Verification of the UPS gene expression from microarray by qPCR analysis. (A) 14 selected genes including Uba1, Ube2r2, Ube3a, Hecw2, Mylip, Nedd4l, Herc1, Rnf144b, Fbxl3, Fbxo31, Cblb, Crbn, Tbl1xr1 and Usp47 were analyzed by qPCR using specific oligonucleotides primers. (B and C) The expressions of standard proteasome catabolic subunits (b1, b2 and b5) and immunoproteasome subunits (b1i, b2i, b5i and PA28a) were detected by qPCR analysis. All data are expressed as means  SE (n = 3 per group). *p < 0.05 versus control.

PARP induced by DOX (Fig. 8E). These results indicate that immunoproteasome catalytic subunits are required for DOXinduced apoptosis. Overexpression of b1i, b2i or b5i reduces DOX-induced the accumulation of polyubiquitinated proteins in cultured NRCMs (Supplementary Fig. 4). Several signalling pathways participate in DOX-induced apoptosis, such as ASK1 and p53, which are known to be degraded by proteasome (Herrmann et al., 2004; Zhang et al., 2009). To detect the effect of b1i, b2i and b5i on the protein levels, we performed western blot analysis. As shown in Fig. 8D, overexpression of b1i, b2i or b5i markedly reduced proapoptotic protein levels of ASK1 and p53 in the heart after DOX treatment (Fig. 8D). Moreover, overexpression of b2i, b5i decreased the mRNA expression of Uba1, Ube2r2, Ube3a, Rnf144b, Fbxl3 and USP47, whereas overexpression of b1i only decreased Ube3a expression (Supplementary Fig. 2). Together, these results indicate that immunoproteasome subunits suppress DOX-induced cardiomyocyte apoptosis partially through enhancing degradation of multiple pro-apoptotic proteins and reversing DOX-induced expression of ubiquitinating enzymes and DUBs.

3.8. Knockdown of immunoproteasome catalytic subunits increases DOX-induced cardiomyocyte apoptosis To further confirm the effect of immunoproteasome catalytic subunits on DOX-induced apoptosis, NRCMs were infected with Ad-shb1i, shb2i, shb5i or Ad-shC and subsequently treated with DOX. TUNEL assay showed that cardiomyocyte apoptosis was increased to approximately 17% by stimulation with DOX, whereas knockdown of b1i, b2i or b5i markedly increased cardiomyocyte apoptosis and caspase-3 activity (Fig. 9A–C). Knockdown of b1i, b2i or b5i further increased the activation of caspase-3 and the cleavage of PARP induced by DOX (Fig. 9E) Moreover, knockdown of b1i, b2i or b5i further up-regulated pro-apoptotic protein levels of ASK1 and p53 in the heart after DOX treatment (Fig. 9D). 4. Disscussion In this study, we identified the genes involved in DOX-induced cardiotoxicity and found 1185 genes were altered. Among them,

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Fig. 4. Effects of DOX on the protein expression of standard and immunoproteasome subunits. Experimental groups were consistent with Fig. 3. (A and B) The protein levels of standard proteasome catalytic subunits (b1, b2 and b5) and immunoproteasome subunits (b1i, b2i, b5i and PA28a) were measured by western blot analysis. Equal protein loading was confirmed by b-actin (left). The intensity of the bands was quantified (right). All data are expressed as means  SE (n = 3 per group). *p < 0.05 versus control.

there were 39 UPS genes, including 1 E1, 3 E2s, 29 E3s, 2 DUBs, 3 immunoproteasome catalytic subunits and 1 proteasome activator. Moreover, DOX markedly reduced proteasome activities and consequently increased the accumulation of polyubiquitinated proteins. Importantly, overexpression of b1i, b2i or b5i significantly inhibited DOX-induced cardiomyocyte apoptosis, and this was partially associated with increased degradation of multiple pro-apoptotic proteins (ASK1 and p53). Knockdown of b1i, b2i or b5i significantly aggravated DOX-induced cardiomyocyte

apoptosis, and this was partially associated with accumulation of multiple pro-apoptotic proteins (ASK1 and p53). UPS plays a key role in the regulation of apoptosis in various heart diseases. Sohns et al. suggested that UPS regulated many apoptotic signal pathways such as the death receptor pathway, the mitochondrial apoptotic pathway, cell cycle regulating pathways and anti-apoptotic regulatory pathways in cardiomyocytes (Sohns et al., 2010). Proteasome degrades polyubiquitinated proteins to regulate protein factors in cells. Bellavista et al. suggested that the

Fig. 5. Effects of DOX on proteasome activities and the level of polyubiquitinated proteins in hearts. Experimental groups were consistent with Fig. 3. (A) The 26S proteasome activities, including the caspase-like, trypsin-like and chymotrypsin-like activities, were measured using specific fluorescent substrates. (B) Total protein extracts were analyzed for ubiquitin conjugates using western blot analysis with an anti-ubiquitin antibody. Equal protein loading was confirmed by b-actin (left). Column graph shows quantification of polyubiquitinated proteins (right). All data are expressed as means  SE (n = 3 per group). *p < 0.05 versus control.

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Fig. 6. Effects of DOX on cardiomyocyte apoptosis in vitro. NRCMs were treated with different doses of DOX (0.5, 1 and 5 mM) for 24 h. (A) Cardiomyocyte apoptosis detected by TUNEL assay. The quantitative analysis of TUNEL-positive nuclei was shown. (B) The expression of Bcl-2 and Bax protein in cardiomyocytes was detected by western blot analysis. Equal protein loading was confirmed by b-actin (top). Column graph showed the quantitative analysis of the ratio of Bax/Bcl-2 (bottom). (C) The activation of caspase-3 and PARP was detected by western blot analysis. Equal protein loading was confirmed by b-actin (left). Column graph showed the quantitative analysis of the expression of cleaved-caspase-3 and cleaved-PARP (right). All data are expressed as means  SE (n = 3 per group). *p < 0.05 versus control.

proteasome which acts as a central core of the UPS is a kind of mandatory terminator of proteins and its inactivation leads to cellular apoptosis (Bellavista et al., 2013). Our results indicated that DOX markedly reduced proteasome activities (Figs. 5A and 7A) and increased the level of polyubiquitinated proteins (Fig. 5B). The proteasome proteolytic activities contain caspase-like, trypsinlike, and chymotrypsin-like peptidase activities within its b-subunits. The three catalytic b-subunits which can exit as constitutive subunits (b1, b2 and b5) and immunosubunits (b1i, b2i and b5i), we then detected the expression of them and found that DOX decreased the expression of immunoproteasome subunits (b1i, b2i and b5i) (Figs. 3C, 4B, 7B and C) but had no effect on standard proteasome subunits (b1, b2 and b5) (Figs. 3B, 4A, and 7D). These results suggest that immunoproteasome might play a critical role in DOX-induced cardiotoxicity. The proteasome assembled with these alternative subunits (b1i, b2i and b5i) is known as the immunoproteasome, which has been reported to be expressed in the heart (Ferrington and Gregerson, 2012). Recent studies suggest that the immunoproteasome possess higher activity to degrade the substrates (Bellavista et al., 2013). Bellavista et al. suggested that selective inhibition of b5i subunit blocks growth and triggers apoptosis in myeloma cell lines and myeloma patient’s primary cells (Bellavista et al., 2013). Ruiz et al. found that inhibition of proteasome induced lymphocyte apoptosis in patients with chronic lymphocytic leukemia (Ruiz

et al., 2006). Apoptosis is a critical pathological process in DOXinduced cardiotoxicity. Both extrinsic death receptor pathway and intrinsic mitochondrial pathway can lead to the activation of caspase-3 (Sohns et al., 2010). Then avtivated caspase-3 hydrolyze PARP between Asp214 and Gly215. The cleaved PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (Oliver et al., 1998). Therefore, dysfunction of immunoproteasome might play a critical role in DOX-induced cardiomyoctye apoptosis. Overexpression of b1i, b2i or b5i significantly attenuated DOXinduced cardiomyocyte apoptosis (Fig. 8A–C and E), while knockdown of b1i, b2i or b5i markedly aggravated DOX-induced cardiomyocyte apoptosis (Fig. 9A–C and E). But inhibition of immunoproteasome genetically did not induce cardiomyocyte apoptosis without DOX treatment (Supplementary Fig. 5). DOX induced the accumulation of polyubiquitinated proteins which could be degraded by proteasome whilst DOX decreased proteasome activities and the expression of immunoproteasome catalytic subunits. Therefore, we demonstrated that the downregulation of immunoproteasome amplify the cardiotoxic effects of polyubiquitinated proteins induced by DOX. Apoptosis could be mediated by many signaling pathways. Activation of ASK1 and p53 has been reported to promote DOX-induced cardiomyocyte apoptosis (Ludke et al., 2012; Sinha et al., 2013; Tokarska-Schlattner et al., 2010; Yi et al., 2006). Our results showed that overexpression of b1i, b2i or

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Fig. 7. Effects of DOX on proteasome activities and the expression of proteasome subunits in cardiomyocytes in vitro. NRCMs were treated with different doses of DOX (0.5, 1 and 5 mM) for 24 h. (A) Caspase-like, trypsin-like and chymotrypsin-like activities were analyzed by specific fluorescent substrates. (B) The qPCR analysis of b1i, b2i, b5i and PA28a mRNA expression was performed. (C) The protein levels of b1i, b2i, b5i and PA28a were detected by western blot analysis. Equal protein loading was confirmed by b-actin (left). The intensity of the bands was quantified (right). (D) The protein levels of standard proteasome catalytic subunits (b1, b2 and b5) were detected by western blot analysis. Equal protein loading was confirmed by b-actin (left). The intensity of the bands was quantified (right). All data are expressed as means  SE (n = 3 per group). *p < 0.05 versus control.

b5i markedly decreased the expression of ASK1 and p53 induced by DOX (Fig. 8D). While knockdown of b1i, b2i or b5i aggravated the expression of ASK1 and p53 (Fig. 9D). We then detected the effects of overexpression of b1i, b2i or b5i on the ubiquitinating enzymes and DUBs which increased in DOX-treated heart to definite whether b1i, b2i or b5i influence other UPS components. The results showed that overexpression of b2i or b5i decreased the mRNA expression of Uba1, Ube2r2, Ube3a, Rnf144b, Fbxl3, and Usp47 and overexpression of b1i only decreased the expression of Ube3a. These results indicated that immunoproteasome not only mediated the DOX-

induced cardiomyocyte apoptosis but also inhibit the expression of many other UPS components. Besides ASK1 and p53 pathway, immunoproteasome could be effective in other pro-apoptotic pathways. Maldonado et al. have demonstrated that knockdown of b1i can prolongate activation of p65 and knockdown b2i and b5i had no effect on delayed termination of p65 (Maldonado et al., 2013). Our results showed that overexpression of b1i had stronger effect on inhibition of apoptosis compared with b2i and it was not consistent with the expression of pro-apoptotic proteins. Hence, it could be supposed that decreased b1i prolongate the activation of p65 while b1i, b2i

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Fig. 8. Overexpression of b1i, b2i or b5i attenuates DOX-induced cardiomyocyte apoptosis and increases pro-apoptotic protein degradation in vitro. NRCMs were infected with Ad-GFP, Ad-b1i, b2i or b5i and then stimulated with DOX (1 mM) for additional 24 h. (A) Representative confocal images of apoptotic nuclei by TUNEL staining (red). Nuclei were counterstained with DAPI (blue). Arrows indicated apoptotic nuclei. (B) Column graph showed the quantitative analysis of TUNEL-positive nuclei. (C) Caspase3 activity was measured by using substrate Ac-DEVD-AFC. (D) Western blot analysis of expression of ASK1 and p53 proteins. Equal protein loading was confirmed by b-actin (left). The intensity of the bands was quantified in the bottom. (E) The activation of caspase-3 and PARP was detected by western blot analysis. Equal protein loading was confirmed by b-actin (left). Column graph showed the quantitative analysis of the expression of cleaved-caspase-3 and cleaved-PARP (right). All data are expressed as means  SE (n = 3 per group). *p < 0.05 versus Ad-GFP. #p < 0.05 versus Ad-GFP + DOX. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

and b5i activate ASK1 and p53 pathway to promote apoptosis in DOX-induced cardiotoxicity. The UPS is the main pathway that promotes the ubiquitination and degradation of intracellular proteins in eukaryotes (Wang and Robbins, 2014). The UPS participates in many biological actions in DOX-induced cardiotoxicity: Yamamoto et al. found DOX increased the protein expression of atrogin-1 which contributed to cardiac

muscle atrophy (Yamamoto et al., 2008). Zhang et al. found that DOX increased the expression of Nrdp1 and Nrdp1 transgenic mice were susceptible to cardiac dysfunction after DOX injection (Zhang et al., 2011). However the DOX-induced proteasome proteolysis activity had been controversial: Liu et al. found that DOX had bidirectional effects on the chymotrypsin-like activity of purified 20S proteasomes (Liu et al., 2008). Kumarapeli et al. established

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Fig. 9. Knockdown of b1i, b2i or b5i increases DOX-induced cardiomyocyte apoptosis and the accumulation of pro-apoptotic proteins. NRCMs were infected with Ad-shC, Ad-shb1i, shb2i or shb5i and then stimulated with DOX (1 mM) for additional 24 h. (A) Representative confocal images of apoptotic nuclei by TUNEL staining (red). Nuclei were counterstained with DAPI (blue). Arrows indicated apoptotic nuclei. (B) Column graph showed the quantitative analysis of TUNEL-positive nuclei (n = 5 per group). (C) Caspase-3 activity was measured by using substrate Ac-DEVD-AFC. (D) Western blot analysis of expression of ASK1 and p53 proteins. Equal protein loading was confirmed by b-actin (left). The intensity of the bands was quantified was shown in the bottom. (E) The activation of caspase-3 and PARP was detected by western blot analysis. Equal protein loading was confirmed by b-actin (left). Column graph showed the quantitative analysis of the expression of cleaved-caspase-3 and cleaved-PARP (right). All data are expressed as means  SE (n = 3 per group). *p < 0.05 versus Ad-shC. #p < 0.05 versus Ad-shC + DOX. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

GFPdgn mice that are suited for monitoring the alterations in proteolytic function of proteasome in the heart and other organs, and demonstrated that DOX enhances UPS-mediated proteolysis in the heart (Kumarapeli et al., 2005). While Sishi et al. revealed that DOX (20 mg/kg) decreased chymotrypsin-like activity in the heart

(Sishi et al., 2013). This paradox are explained methodologically (Kumarapeli et al., 2005) or dose dependently (Dimitrakis et al., 2012) or time dependently. We prefer to believe that DOX dosedependently induce dysfunction of proteasomes, because our results revealed that DOX not only decreased the proteasome

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activities, but also decreased the mRNA and protein expression of immunoproteasome catalytic subunits (b1i, b2i, b5i) in the DOXtreated heart and cultured NRCMs detected by qPCR and western blot analysis (Figs. 3C, 4B, 6D and E). In conclusion, this study identified 39 UPS genes in DOXinduced cardiotoxicity, in which the immunoproteasome subunits were shown to regulate other UPS gene expression and cardiomyocyte apoptosis, and the underlying mechanisms were partially associated with decreased degradation of several pro-apoptotic proteins (such as ASK1 and p53). Thus, immunoproteasomemediated proteolysis has emerged as an important molecular mechanism for regulating DOX-induced cardiomyocyte apoptosis, and may be a novel therapeutic target for prevention of DOXinduced cardiotoxicity. However, it will be important to investigate the role of other UPS gene members and the underlying causal mechanisms in DOX-induced cardiotoxicity. The immunoproteasome is a double-edged sword in various tumor developments. The specific inhibition of b5i (PR-957) prohibit growth and induced apoptosis in cultured primary multiple myeloma (MM) patient tumor cells and MM cell lines. The specific inhibition of b1i (UK-101) prohibit the growth activity in prostatic cancer cell. While renal carcinoma cells, hepatocellular carcinoma cell lines and malignant melanoma cells which expressed lower levels of b1i and b5i are susceptible to metastatic lesions. The mice which are lacking b1i prefer to develop spontaneous uterine leiomyosarcoma (Bellavista et al. 2013). Therefore, we need to evaluate the expression of immunoproteasome in primary tumor when we target immunoproteasome to ameliorate DOX-induced cardiotoxicity. Conflict of interest There are no conflicts of interest. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant numbers 81025001, 81330003 and 510025); the 973 Program (2012CB517802) and Chang Jiang Scholar Program; Beijing Key Laboratory Special Program (PXM2015_014226_000062).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tox.2015.04.009.

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