ELSEVIER
Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet
Toxicology Letters
TL
Cytotoxicity and gene expression profiling of two hydroxylated polybrominated diphenyl ethers in human H295R adrenocortical carcinoma cells
Renfang Song a, b,c,1, Tiago L. Duartea,d,1, Gabriela M. Almeida a,e, Peter B. Farmerª, Marcus S. Cooke a,f, Wenbing Zhangb, Guoying Shengb, Jiamo Fub, George D.D. Jonesª,*
a Department of Cancer Studies & Molecular Medicine, University of Leicester, University Road, Leicester LE1 7RH, UK
b State Key Laboratory of Organic Geochemistry, Guangzhou Research Center of Mass Spectrometry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou, China
” Graduate School of the Chinese Academy of Sciences, Beijing, China
d IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
e Institute of Molecular Pathology & Immunology, University of Porto, Porto, Portugal
f Department of Genetics, University of Leicester, UK
ARTICLE INFO
Article history: Received 10 September 2008 Received in revised form 18 November 2008 Accepted 20 November 2008 Available online 27 November 2008
Keywords: OH-PBDE H295R adrenocortical carcinoma cells Toxicity Endoplasmic reticulum stress Unfolded protein response
ABSTRACT
Polybrominated diphenyl ethers (PBDEs) are commonly used as flame retardants in a variety of com- mercial and household products. They have been detected in the environment and accumulate in mammalian tissues and fluids. PBDE toxicity is thought to be associated with endocrine disruption, developmental neurotoxicity and changes in fetal development. Although humans are exposed to PBDEs, our knowledge of the effects of PBDE metabolites on human cells with respect to health risk is insuffi- cient. Two hydroxylated PBDEs (OH-PBDEs), 2-OH-BDE47 and 2-OH-BDE85, were investigated for their effects on cell viability/proliferation, DNA damage, cell cycle distribution and gene expression profiling in H295R adrenocortical carcinoma cells. We show that the two agents are cytotoxic in a dose-dependent manner only at micromolar concentrations, with 2-OH-BDE85 being more toxic than 2-OH-BDE47. However, no DNA damage was observed for either chemical, suggesting that the biological effects of OH- PBDEs occur primarily via non-genotoxic routes. Furthermore, no evidence of aryl hydrocarbon receptor (AHR)-mediated, dioxin-like toxicity was observed. Instead, we report that a micromolar concentra- tion of OH-PBDEs induces transcriptional changes associated with endoplasmic reticulum stress and the unfolded protein response. We discuss whether OH-PBDE bioaccumulation could result in impairment of the adrenocortical secretory function.
@ 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Polybrominated biphenyl ethers (PBDEs) are synthetic com- pounds used as flame retardants in a variety of construction materials, textiles and polymers for electronic equipment. PBDEs are hydrophobic, stable and persist in the environment (Hites, 2004). PBDEs and their metabolites, hydroxylated PBDEs (OH- PBDEs), have been found in blood, adipose tissues and liver of fish, birds and mammals (Teuten et al., 2005; Wolkers et al., 2004), as well as in human blood, serum, adipose tissue, breast milk, placen-
tal tissue and the brain (Siddiqi et al., 2003; Athanasiadou et al., 2008; Domingo et al., 2008). Importantly, concentrations of PBDEs in breast milk (Hooper and McDonald, 2000; Kodavanti and Derr- Yellin, 2002; Hites, 2004) or blood (Schecter et al., 2005) increased exponentially in the last decades. Although in some cases human exposure has decreased in recent years (e.g. Lind et al., 2003; Domingo et al., 2008; Polder et al., 2008), PBDEs still present a potential risk to both the environment and to public health.
PBDEs have a similar chemical structure to other environmen- tal, persistent, organic pollutants such as polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Exposure to PCBs is associated with adverse effects on human health. PCBs are complete carcinogens in rodents and display genotoxic activ- ity in vitro and in vivo (Safe, 1984; Silberhorn et al., 1990). PBDEs and their metabolites are thought to be toxic to humans, but little is known about their mechanisms of toxicology. PBDEs cause develop- mental neurotoxicity in rodents (Viberg et al., 2003), which could be related to the high content of PBDEs in the brain (Naert et al., 2007) and in breast milk, from where it is transferred to nursing infants
Abbreviations: AHR, aryl hydrocarbon receptor; ERAD, ER-associated degra- dation; ER, endoplasmic reticulum; PBDE, polybrominated diphenyl ether; PCB, polychlorinated biphenyl; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; UPR, unfolded protein response.
* Corresponding author. Tel .: +44 116 223 1841; fax: +44 116 223 1840. E-mail address: gdj2@le.ac.uk (G.D.D. Jones).
1 Both authors contributed equally to this work.
who are in the early stages of brain development (Coburn et al., 2007). There is increasing concern about the potential endocrine- disrupting properties of brominated flame retardants and their derivatives, which could affect steroidogenic enzymes responsible for the biosynthesis of various steroid hormones, and eventually cause reproductive problems, cancer and other pathological con- ditions related to growth and development. In this respect, some PBDEs were determined to have endocrine-disrupting effects in vitro (Meerts et al., 2001) and in vivo (Bonefeld-Jorgensen et al., 2006; Hardell et al., 2006). OH-PBDE congeners have structural sim- ilarities with thyroid hormones and bind human thyroid hormone receptors and serum thyroid hormone binding proteins (Siddiqi et al., 2003). OH-PBDEs also affect the activity of two key enzymes in the production of estrogens and androgens, aromatase (CYP19) and CYP17, in H295R adrenocortical carcinoma cells at concentrations in the low micromolar range (between 0.5 and 10 p.M) (Canton et al., 2005, 2006; He et al., 2008a). Moreover, certain OH-PBDEs are able to bind to cytosolic aryl hydrocarbon receptors (AHR), becoming AHR agonists or antagonists (Chen and Bunce, 2003).
Although human tissues contain measurable levels of PBDEs, lit- tle is known about the toxicological effects of PBDE metabolites in humans (Darnerud et al., 2005). Adrenal glands, which have massive blood flow and high lipid content, represent a major accu- mulation site for dioxins, PCBs and PBDEs in the body. Recent work has shown that PBDEs and their derivatives affect adrenal steroido- genesis in H295R adrenocortical carcinoma cells (Canton et al., 2005, 2006; He et al., 2008a), but information on their toxicities and mechanisms of action is limited. In this work, we evaluated the effects of two environmental PBDE metabolites (2-OH-BDE47 and 2-OH-BDE85) on DNA damage, cell proliferation, cell cycle dis- tribution and gene expression profiling in H295R adrenocortical carcinoma cells. BDE47 is one of the most widespread and per- sistent PBDE congeners in the environment (Law et al., 2003). In vertebrates, it undergoes phase I metabolism leading to the forma- tion of OH-BDE47 (Qiu et al., 2007). BDE85, on the other hand, has a similar structure to TCDD and affinity for the AHR (Chen et al., 2001).
2. Materials and methods
2.1. Chemicals and reagents
2-OH-BDE47 and 2-OH-BDE85 were synthesized and provided by Dr. Michael H.W. Lam, Department of Biology and Chemistry, City University of Hong Kong, Hong Kong. All other chemicals, tissue culture media and reagents were purchased from Sigma (Poole, UK) unless otherwise stated.
2.2. Cell culture and chemical exposure
The H295R adrenocortical carcinoma cell line was obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium sup- plemented with Ham’s nutrient mixture F-12 with 1% Insulin Transferrin Selenium (ITS) Premix and 2.5% Nu-Serum at 37 ℃ in a 5% CO2 atmosphere. Cells were exposed to chemicals dissolved in 0.1% dimethyl sulfoxide (DMSO) (final concentration) for different incubation periods. Control cells were incubated with the vehicle (DMSO) only.
2.3. Single cell gel electrophoresis (comet assay)
DNA damage was measured with the alkaline comet assay, which is a straight- forward and sensitive method for measuring a variety of DNA lesions in individual cells (Singh et al., 1988), and expressed as the percentage of tail DNA, as described (Duarte et al., 2007).
2.4. Cell proliferation assay
Cell proliferation was studied by using the CyQUANT NF cell proliferation assay (Invitrogen, California, USA), which measures cellular DNA content via fluorescent dye binding, according to the manufacturers’ instructions. Briefly, 10,000 cells in 100 ul were seeded in a 96-well plate and left to attach for 24h. Cells were then treated with different concentrations of chemicals in 100 pl of medium for 24h. The agent-containing medium was removed and 100 ul of dye binding solution directly added and incubated for 60 min. Fluorescence was recorded using a FLU- Ostar OPTIMA fluorescence microplate reader (Offenburg, Germany) with excitation at 480 nm and emission detection at 520 nm.
2.5. Cell cycle analysis
Cells were plated in 6-well plates at 100,000 cells per well. After 24 h, cells were treated with test agents or vehicle (control) for up to 72 h. Cells were harvested at each incubation period of interest, fixed in 70% ethanol and subsequently resus- pended in PBS containing 0.1 mg/ml RNase A and 5 µg/ml propidium iodide, prior to analysis. Cellular DNA content was analysed using a Becton Dickinson FACScan flow cytometer and CellQuest software (Becton Dickinson, San Jose, CA), plotting 10,000 events per sample. Cell cycle distribution data analysis was subsequently performed using ModFit LT software (Becton Dickinson, Cowley, UK).
2.6. Statistical analysis
For the above Comet, cell proliferation and cell cycle assays, statistical analysis was performed with SPSS14 (SPSS Inc., Chicago, Illinois, USA). Statistically significant differences from control were calculated by a one-way ANOVA followed by the post hoc Tukey’s test.
2.7. Microarray analysis
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, UK) and 5 µg were reverse transcribed using the One-cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, California, USA). Biotin-labeled cRNA was synthesised by in vitro transcription with the Affymetrix IVT labeling kit. Fragmented, biotin-labeled CRNAs (10 µg) were hybridised to GeneChip Affymetrix Human U133 Plus 2.0 arrays and these were washed and stained according to Affymetrix EukGE-WS2v5 Gene Chip protocol. Probe arrays were scanned at 570 nm using an Affymetrix GeneChip scanner and the fluorescence intensity of the scanned image registered in CEL intensity files. The data were deposited in NCBI’s Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE8588. DNA-Chip Analyser (dChip) software (Li and Wong, 2001) was used to normalise the fluorescence intensity over multiple arrays, to calculate model-based expression values from the differences between perfect match and mismatch probes in the cell intensity files and to filter a set of 2-OH-BDE47- and 2-OH-BDE85-responsive genes for which the expression levels were significantly altered in three independent experiments (paired t-test, P<0.05), with an average fold change ≥3 or ≤-3 when comparing with the respective control. In addition, the software was employed to determine the probability of having a particular number of genes of a certain Gene Ontology (GO) biological process in the generated list of filtered genes, using hypergeometric distribution; GO enrichment was assumed at P<0.001.
| Gene name | Forward primer (5'-3') | Reverse primer (5'-3') | Product length (bp) |
|---|---|---|---|
| ATF3 | ATGGGCAGTATGATGGCAGGTCC | TGAGTGGAGAGTCCTAACCCTTTGGA | 101 |
| DDIT3 | AGAGCCCTCACTCTCCAGATTCCA | TCTGTTTCCGTTTCCTGGTTCTCC | 90 |
| HSPA5 | AAGCCACCAAGATGCTGACATTGA | GCCTGCACTTCCATAGAGTTTGCTG | 105 |
| TIPARP | AATGGAGGCTGTGAGCAATGTGAG | CTTTGGACAGGGAATACGCATGG | 95 |
| TRIB3 | GCTGCCAACAGTGGATTGAGTTTG | GGCTTCTGCCTTTCTCCCTTCTG | 103 |
| CYP1B1 | ACAACTAACGCAACCAAGTGTGCT | GAAACCCTGCTTCATTTCCATGTC | 83 |
| ADRA2A | TTTCTGCCTCACATCAGCCCTGT | GGGAGAGAGGGAACACTGGAAAGAG | 105 |
| SREBF1 | CTATGTGGCGGCTGCATTGAGAGT | GCACTGCTCAGGAAGAAGCGTGTC | 81 |
| GADD45xx | AGGATTACAGAAACTGATGCCAAGGG | TCCATCTGCAAAGTCATCTATCTCCG | 85 |
| HPRT1 | GCAGACTTTGCTTTCCTTGGTCAG | GTCTGGCTTATATCCAACACTTGGTG | 103 |
2.8. Real-time RT-PCR
Real-time reverse transcription-PCR (RT-PCR) was performed as described (Duarte and Jones, 2007), with the following modifications. The reaction mix consisted of Brilliant SYBRGreen QPCR Master Mix (Stratagene), 2.5 mM MgCl2, 150 nM each primer and 1 pl of cDNA. Primer sequences are shown in Table 1. The amplification protocol consisted of a denaturation step at 95 ℃ for 10 min and 40 cycles of 95 ℃ for 30 s, 57 ℃ for 1 min and 72 ℃ for 1 min.
3. Results and discussion
3.1. Cytotoxicity of OH-PBDES
The effects of the two OH-PBDEs on H295R cell prolifera- tion were evaluated after 24-h exposure by comparison with untreated, control cells (Fig. 1). At concentrations below 10 µM, both OH-PBDEs inhibited H295R cell proliferation, with only 2-OH-BDE85 causing a slight reduction in cell number when compared to the number of cells at 0h. From 10 pM onwards both compounds induced cell death in a concentration-dependent manner, with 2-OH-BDE85 clearly being the more cytotoxic of the two.
3.2. Cell cycle effects of OH-PBDEs
Flow cytometric analysis of cellular DNA content was subse- quently performed to determine whether the cytotoxic effects of the two OH-PBDEs were accompanied by cell cycle arrest at spe- cific checkpoints. Cell cycle distribution changes were monitored in H295R cells following exposure to 10 p.M 2-OH-BDE47 or 2-OH- BDE85 over 24-72 h. In the controls, the percentage of cells in the Go/G1, S and G2/M phases of the cell cycle was 60-70%, 20% and 10-20%, respectively. Notably, both compounds (10 µM) caused a significant decrease in the percentage of G2/M cells and concomi- tant increase in G0/G1 cells at 24 h of exposure (Fig. 2); however, there was no significant difference noted between the exposed and control cells at 48 and 72 h.
3.3. Genotoxicity of OH-PBDES
Barber et al. (2006) reported increased chromosomal damage in the micronucleus test in MCF7 cells treated with low-dose PBDEs. Recent reports showed that incubation of human neuroblastoma cells with the parental compound BDE-47 induces genotoxicity and apoptotic cell death in a dose-dependent manner (He et al., 2008b),
100
Relative cell numbers (%)
2-OH-BDE47
80
2-OH-BDE85
60
40
20
0
0
2
5
10
20
50
Concentration (uM)
Relative cell numbers (%)
100
☐
G0/G1
80
*
*
☒
S
60
☐ G2/M
40
20
5
*
0
24h
48h
72h
24h
48h
72h
24h
48h
72h
Control
2-OH-BDE47
2-OH-BDE85
through the formation of reactive oxygen species (Gao et al., 2008). To investigate whether the anti-proliferative effects of OH-PBDEs were produced via a genotoxic route, we employed the alkaline comet assay. We exposed H295R cells to different concentrations of 2-OH-BDE47 or 2-OH-BDE85 for 12 h. In the assay, DNA lesions including single strand breaks, double strand breaks and alkali labile sites are detected as an increase in the percentage of DNA
(A)
50
-4-2-OH-BDE47
40
-0-2-OH-BDE85
% Tail DNA
30
20
10
0
0
2
5
10
20
Concentration (uM)
(B) 50
-o-C
40
-.- C+
-+P
% Tail DNA
-+-P+
30
0-2-OH-BDE85
2-OH-BDE85+
+
20
10
0
0.25
0.5
1
3
24
Time (h)
in the ‘tail’ of the comets. However, we found no prominent tail formation in the treated samples, which was no different from the control (Fig. 3A). Even at a cytotoxic OH-PBDE concentration (20 µM) there was no significant formation of DNA damage compared to control (P>0.05; ANOVA). We could not exclude, however, the possibility that cells repaired any DNA damage formed during the incubation period. Therefore, in a subsequent experiment we compared the DNA damage accumulation in the presence or absence of two DNA repair inhibitors, hydroxyurea and cytosine arabinoside, which inhibit DNA synthesis during excision repair (Martin et al., 1999). Fig. 3B shows the accumulation of DNA damage when H295R cells were incubated with 30 uM hydrogen peroxide (positive control) or 10 M 2-OH-BDE85 over a 24h time period. When cells were incubated with 10 p.M 2-OH-BDE85 in the presence of the DNA repair inhibitors the slight increase in tail DNA observed at 24 h was not greater than that observed in control cells. Similar results were obtained when cells were incubated with 10 µM 2-OH-BDE47 (Supplementary Fig. 1). Importantly, this modified version of the assay also allows the detection of DNA strand breaks and alkali labile sites generated by the excision repair of most DNA lesions. Hence our data suggest that the toxicity of OH-PBDEs in H295R cells cannot be explained by a genotoxic mechanism.
3.4. Gene expression profiling
To investigate the toxicological action of the two OH-PBDEs, we incubated H295R cells with 10 p.M 2-OH-BDE47 or 2-OH-BDE85 for 24 h, a treatment that severely inhibited cell proliferation (Fig. 1), and examined changes in global mRNA profiles using Affymetrix high-density oligonucleotide arrays. Experiments were performed on three separate occasions and, in each experiment, control cells were incubated with vehicle (DMSO) for the same period of time. The HG-U133 Plus 2.0 arrays contain probes for over 47,000 tran- scripts and variants. A total of 430 annotated genes and 152 genes or expressed sequence tags with unknown function had their expres- sion significantly (P<0.05) altered by at least 3-fold after exposure to either 2-OH-BDE47 or 2-OH-BDE85 (Supplementary data Table 1). Despite the higher number of significant changes induced by 2-OH-BDE85 (312 annotated and 104 unknown genes vs. 208 anno- tated and 67 unknown genes for 2-OH-BDE47), it was apparent from the two-way hierarchical clustering analysis of the filtered genes and samples according to their patterns of gene expres- sion that both compounds induced similar expression profiles in H295R cells (Fig. 4). The gene expression changes induced by both compounds could be classified into five significantly enriched Gene Ontology biological processes: response to unfolded protein, apoptosis, cell cycle, transcription and organic acid metabolism (Table 2).
Given the analogous chemical structures, it has been anticipated that PBDEs could have similar toxicological properties to TCDD, whose toxic effects are mostly due to alterations in gene expres- sion mediated by the transcription factor AHR. In this study, whilst we observed the induction of 3 dioxin-responsive genes (AHR, CYP1B1 and TIPARP), we did not observe changes in the expres- sion of many well established AHR-responsive genes like CYP1A1, CYP1A2, NAD(P)H dehydrogenase, quinone 1, UGT1A1/6, aldehyde dehydrogenase family 3, member A1 or glutathione S-transferase Ya (Fletcher et al., 2005). Our results with 2-OH-BDE47 and 2- OH-BDE85 agree with previous observations that both parental compounds (BDE47 and BDE85) have a negligible effect on AHR binding to the dioxin response element and on CYP1A1 and CYP1B1 expression or activity (Chen and Bunce, 2003; Peters et al., 2004; Sanders et al., 2005).
On the other hand, we show that OH-PBDE-mediated cytotox- icity in H295R cells is accompanied by the induction of genes
Control III Control I
Control II
2-OH-BDE47 III
2-OH-BDE47 1
2-OH-BDE47 II
2-OH-BDE85 III
2-OH-BDE85 1
2-OH-BDE85 II
involved in endoplasmic reticulum (ER) stress response, includ- ing those that mediate correct protein folding, cell cycle arrest and cell death/apoptosis. Adrenocortical cells express several path- ways of secretory protein biosynthesis and the ER is the cellular compartment where these proteins are synthesised and correctly folded, with the assistance of molecular chaperones and folding enzymes. Unfolded/mis-folded proteins are retained in the ER, translocated back to the cytoplasm by the ER-associated degrada- tion (ERAD) machinery, and degraded by the ubiquitin-proteasome
| Probe set ID | Gene ID1 | Gene title | Fold change | |
|---|---|---|---|---|
| 2-OH-BDE472 | 2-OH-BDE852 | |||
| Response to unfolded protein | ||||
| 226665_at | 130872 | Activator of heat shock 90kDa protein ATPase homolog 2 (AHSA2) | 3.4 | |
| 202842_s_at | 4189 | DNAJ (Hsp40) homolog, subfamily B, member 9 (DNAJB9) | 4.3 | 6.1 |
| 208499_s_at | 5611 | DNAJ (Hsp40) homolog, subfamily C, member 3 (DNAJC3) | 3.5 | |
| 218696_at | 9451 | Eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) | 3.5 | |
| 241716_at | 3329 | Heat shock 60kDa protein 1 (HSPD1) | 8.4 | |
| 202581_at | 3304 | Heat shock 70kDa protein 1B (HSPA1B) | -4.1 | -5.0 |
| 211538_s_at | 3306 | Heat shock 70kDa protein 2 (HSPA2) | 4.3 | |
| 230031_at | 3309 | Heat shock 70kDa protein 5 (HSPA5/GRP78) | 4.5 | 5.1 |
| 1569380_a_at | 9709 | Homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERPUD1) | 6.4 | |
| Apoptosis | ||||
| 210538_s_at | 330 | Baculoviral IAP repeat-containing 3 (BIRC3) | 26.1 | |
| 1557257_at | 8915 | B-cell CLL/lymphoma 10 (BCL10) | 5.3 | |
| 207829_s_at | 662 | BCL2/adenovirus E1B 19kDa interacting protein 1 (BNIP1) | 3.0 | |
| 209406_at | 9532 | BCL2-associated athanogene 2 (BAG2) | 6.8 | |
| 224414_s_at | 84674 | Caspase recruitment domain family, member 6 (CARD6) | -6.5 | |
| 203989_x_at | 2149 | coagulation factor II (thrombin) receptor (F2R) | -3.4 | |
| 209383_at | 1649 | DNA-damage-inducible transcript 3 (DDIT3/CHOP) | 13.6 | 13.7 |
| 202887_s_at | 54541 | DNA-damage-inducible transcript 4 (DDIT4) | 3.4 | |
| 212374_at | 10116 | Fem-1 homolog b (C. elegans) (FEM1B) | 3.1 | |
| 210655_s_at | 2309 | Forkhead box O3A (FOXO3) | 3.2 | |
| 206865_at | 8739 | Harakiri, BCL2 interacting protein (HRK) | 5.8 | |
| 231733_at | 59082 | ICEBERG caspase-1 inhibitor (ICEBERG) | 3.6 | 5.4 |
| 226352_at | 133746 | Junction-mediating and regulatory protein (JMY) | 3.2 | 4.1 |
| 209193_at | 5292 | Pim-1 oncogene (PIM1) | 4.8 | |
| 227751_at | 9141 | Programmed cell death 5 (PDCD5) | 3.8 | 5.8 |
| 202014_at | 23645 | Protein phosphatase 1, regulatory (inhibitor) subunit 15A (PPP1R15A/GADD34) | 6.0 | 9.7 |
| 212884_x_at | 10452 | Translocase of outer mitochondrial membrane 40 homolog (TOMM40) | 3.7 | |
| 1555788_a_at | 57761 | Tribbles homolog 3 (Drosophila) (TRIB3) | 10.2 | 10.4 |
| Cell cycle | ||||
| 1555785_a_at | 54464 | 5'-3' exoribonuclease 1 (XRN1) | 3.4 | |
| 227896_at | 56647 | BRCA2 and CDKN1A interacting protein (BCCIP) | -4.3 | |
| 1556006_s_at | 1452 | Casein kinase 1, alpha 1 (CSNK1A1) | 3.3 | |
| 214683_s_at | 1195 | CDC-like kinase 1 (CLK1) | 6.7 | |
| 217010_s_at | 995 | Cell division cycle 25C (CDC25C) | -3.8 | |
| 217988_at | 57820 | Cyclin B1 interacting protein 1 (CCNB1IP1) | 3.2 | |
| 204826_at | 899 | Cyclin F (CCNF) | -5.3 | -5.8 |
| 220046_s_at | 57018 | Cyclin L1 (CCNL1) | 4.0 | |
| 206967_at | 904 | Cyclin T1 (CCNT1) | 3.4 | |
| 210021_s_at | 10309 | Cyclin U (CCNU/UDG2) | 3.7 | |
| 236313_at | 1030 | Cyclin-dependent kinase inhibitor 2B (CDKN2B/p15) | -3.1 | |
| 204015_s_at | 1846 | Dual specificity phosphatase 4 (DUSP4) | 10.3 | |
| 217370_x_at | 2521 | Fusion (involved in t(12;16) in malignant liposarcoma) (FUS) | 3.7 | 4.3 |
| 203725_at | 1647 | Growth arrest and DNA-damage-inducible, alpha (GADD45A) | 8.9 | 8.2 |
| 1560402_at | 60674 | Growth arrest-specific 5 (GAS5) | 3.9 | 3.6 |
| 202934_at | 3099 | Hexokinase 2 (HK2) | 4.4 | 4.5 |
| 207361_at | 26959 | HMG-box transcription factor 1 (HBP1) | 7.2 | |
| 227013_at | 26524 | LATS, large tumor suppressor, homolog 2 (Drosophila) (LATS2) | 3.2 | |
| 203036_s_at | 9788 | Metastasis suppressor 1 (MTSS1) | 3.4 | |
| 221527_s_at | 56288 | Par-3 partitioning defective 3 homolog (C. elegans) (PARD3) | 3.6 | |
| 202883_s_at | 5519 | Protein phosphatase 2, regulatory subunit A (PR 65), beta (PPP2R1B) | -3.2 | |
| 200730_s_at | 7803 | Protein tyrosine phosphatase type IVA, member 1 (PTP4A1) | 3.3 | |
| 204346_s_at | 11186 | Ras association (RalGDS/AF-6) domain family 1 (RASSF1) | 4.1 | |
| 237626_at | 9821 | RB1-inducible coiled-coil 1 (RB1CC1) | 4.5 | |
| 223195_s_at | 83667 | Sestrin 2 (SESN2) | 5.5 | 6.4 |
| 220961_s_at | 9238 | Transforming growth factor beta regulator 4 (TBRG4) | -3.8 | |
| 225610_at | 115426 | Ubiquitin-like, containing PHD and RING finger domains, 2 (UHRF2) | 3.0 | |
| Transcription | ||||
| 202672_s_at | 467 | Activating transcription factor 3 (ATF3) | 11.8 | 18.0 |
| 238476_at | 153222 | Adult retina protein (LOC153222) | 3.9 | 6.1 |
| 202820_at | 196 | Aryl hydrocarbon receptor (AHR) | 3.7 | |
| 201170_s_at | 8553 | Basic helix-loop-helix domain containing, class B, 2 (BHLHB2) | 3.6 | 8.8 |
| 221530_s_at | 79365 | Basic helix-loop-helix domain containing, class B, 3 (BHLHB3) | 3.2 | |
| 235965_at | 168620 | Basic helix-loop-helix domain containing, class B, 8 (BHLHB8) | 25.1 | |
| 1558560_s_at | 8548 | Basic leucine zipper nuclear factor 1 (JEM-1) | 3.1 | |
| 228289_at | 29117 | Bromodomain containing 7 (BRD7) | -3.6 | -4.6 |
| 209357_at | 10370 | Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 (CITED2) | 3.1 | |
| 203973_s_at | 1052 | CCAAT/enhancer binding protein (C/EBP), delta (CEBPD) | 4.4 | 4.1 |
| 204203_at | 1054 | CCAAT/enhancer binding protein (C/EBP), gamma (CEBPG) | 4.5 | 6.7 |
| 202979_s_at | 58487 | CREB/ATF bZIP transcription factor (CREBZF) | 3.2 | |
| 219947_at | 50856 | C-type lectin domain family 4, member A (CLEC4A) | 5.4 | 5.7 |
| Probe set ID | Gene ID1 | Gene title | Fold change | |
|---|---|---|---|---|
| 2-OH-BDE472 | 2-OH-BDE852 | |||
| 229069_at | 84324 | Cytokine induced protein 29 kDa (CIP29) | 3.6 | 3.2 |
| 210269_s_at | 8227 | DNA segment on chromosome X and Y (unique) 155 expressed sequence, isoform 1 (SFRS17A) | 4.3 | |
| 227404_s_at | 1958 | Early growth response 1 (EGR1) | 3.8 | 6.6 |
| 222869_s_at | 55520 | elaC homolog 1 (E. coli) (ELAC1) | -3.1 | |
| 230102_at | 2119 | Ets variant gene 5 (ETV5) | 5.0 | |
| 1569076_a_at | 162962 | FLJ16287 protein | 4 | |
| 206912_at | 2304 | Forkhead box E1 (thyroid transcription factor 2) (FOXE1) | -3.8 | |
| 225262_at | 2355 | FOS-like antigen 2 (FOSL2) | 3.5 | |
| 202949_s_at | 2274 | Four and a half LIM domains 2 (FHL2) | 3.4 | 3.4 |
| 204755_x_at | 3131 | Hepatic leukemia factor (HLF) | 5.3 | |
| 213359_at | 3184 | Heterogeneous nuclear ribonucleoprotein D (HNRPD) | 5.2 | 12 |
| 201209_at | 3065 | Histone deacetylase 1 (HDAC1) | -3.1 | |
| 223908_at | 55869 | Histone deacetylase 8 (HDAC8) | -3.3 | |
| 203543_s_at | 687 | Kruppel-like factor 9 (KL9) | 4.7 | 8.1 |
| 203004_s_at | 4209 | MADS box transcription enhancer factor 2, polypeptide D (MEF2D) | 3.9 | |
| 228846_at | 4084 | MAX dimerization protein 1 (MXD1) | 3.8 | |
| 203238_s_at | 4854 | Notch homolog 3 (Drosophila) (NOTCH3) | -3.6 | |
| 208003_s_at | 10725 | Nuclear factor of activated T-cells 5 (NFAT5) | 3.2 | |
| 228388_at | 4793 | Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, beta (NFKBIB) | 3.8 | |
| 203574_at | 4783 | Nuclear factor, interleukin 3 regulated | 4.9 | 9.3 |
| 234471_s_at | 57727 | Nuclear receptor coactivator 5 (NCOA5) | -3.5 | |
| 202340_x_at | 3164 | Nuclear receptor subfamily 4, group A, member 1 (NR4A1) | 4.3 | |
| 216248_s_at | 4929 | Nuclear receptor subfamily 4, group A, member 2 (NR4A2) | 5.8 | |
| 227220_at | 152518 | Nuclear transcription factor, X-box binding-like 1 (NFXL1) | 3.0 | |
| 202861_at | 5187 | Period homolog 1 (Drosophila) (PER1) | 4.8 | |
| 203057_s_at | 7799 | PR domain containing 2, with ZNF domain (PRDM2) | -4.04 | |
| 243624_at | 9063 | Protein inhibitor of activated STAT, 2 (PIAS2) | -3.4 | 3.2 |
| 210191_s_at | 10745 | Putative homeodomain transcription factor 1 (PHTF1) | -3.9 | -3.1 |
| 210479_s_at | 6095 | RAR-related orphan receptor A (RORA) | 4.2 | |
| 232971_at | 64783 | RNA binding motif protein 15 (RBM15) | 4.0 | |
| 206554_x_at | 6419 | SET domain and mariner transposase fusion gene (SETMAR) | -3.3 | |
| 202308_at | 6720 | Sterol regulatory element binding transcription factor 1 (SREBF1) | -6.5 | |
| 207678_s_at | 11063 | SRY (sex determining region Y)-box 30 (SOX30) | 3.9 | 3.9 |
| 215048_at | 140883 | Suppressor of hairy wing homolog 2 (Drosophila) (SUHW2) | 3.4 | |
| 1558208_at | 23435 | TAR DNA binding protein (TARDBP) | 6.2 | |
| 242243_at | 7110 | TATA element modulatory factor 1 (TMF1) | 4.1 | |
| 223393_s_at | 57616 | Teashirt family zinc finger 3 (TSHZ3) | -3.1 | |
| 236978_at | 9969 | Thyroid hormone receptor associated protein 1 (THRAP1) | -3.3 | |
| 232097_at | 9878 | TOX high mobility group box family member 4 (TOX4) | 3.3 | |
| 242538_at | 7027 | Transcription factor Dp-1 (TFDP1) | -3.7 | |
| 36711_at | 23764 | v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (MAFF) | 5.9 | 15.9 |
| 238496_at | 54904 | Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1) | 4.0 | |
| 200670_at | 7494 | X-box binding protein 1 (XBP1) | 6.3 | 6.7 |
| 222863_at | 65986 | Zinc finger and BTB domain containing 10 (ZBTB10) | 5.4 | |
| 226284_at | 57621 | Zinc finger and BTB domain containing 2 (ZBTB2) | 4.7 | |
| 1554045_at | 7572 | Zinc finger protein 24 (ZNF24) | 8.0 | |
| 219595_at | 7574 | Zinc finger protein 26 (ZNF26) | 5.3 | |
| 228711_at | 7587 | Zinc finger protein 37A (ZNF37A) | -3.0 | |
| 236429_at | 55769 | Zinc finger protein 83 (ZNF83) | -4.6 | |
| 1569241_a_at | 81931 | Zinc finger protein 93 (ZNF93) | 3.6 | 3.9 |
| 216960_s_at | 7692 | Zinc finger protein 133 (ZNF133) | 3.4 | |
| 215887_at | 11179 | Zinc finger protein 277 pseudogene (ZNF277P) | 4.2 | |
| 225539_at | 49854 | Zinc finger protein 295 (ZNF295) | 4.0 | |
| 229136_s_at | 55900 | Zinc finger protein 302 (ZNF302) | 4.2 | |
| 1554985_at | 252884 | Zinc finger protein 396 (ZNF396) | 6.8 | 3.9 |
| 219848_s_at | 9668 | Zinc finger protein 432 (ZNF432) | 3.1 | 3.2 |
| 233175_at | 10794 | Zinc finger protein 460 (ZNF460) | 3.9 | |
| 217593_at | 65982 | Zinc finger protein 447 (ZNF447) | 3.5 | |
| 229901_at | 118738 | Zinc finger protein 488 (ZNF488) | -3.8 | -5.3 |
| 228099_at | 162972 | Zinc finger protein 550 (ZNF550) | 3.4 | |
| 211721_s_at | 90233 | Zinc finger protein 551 (ZNF551) | -3.5 | |
| 224518_s_at | 84527 | Zinc finger protein 559 (ZNF559) | -3.3 | |
| 242028_at | 163050 | Zinc finger protein 564 (ZNF564) | -3.3 | |
| 1570038_at | 152687 | Zinc finger protein 595 (ZNF595) | 5.2 | |
| 239482_x_at | 7562 | Zinc finger protein 708 (ZNF708) | -5.5 | |
| 238907_at | 284323 | Zinc finger protein 780A (ZNF780A) | -5.6 | |
| Organic acid metabolism | ||||
| 201000_at | 16 | Alanyl-tRNA synthetase (AAR) | 3.6 | |
| 203946_s_at | 384 | Arginase, type II (ARG2) | 4.6 | 3.9 |
| 205047_s_at | 440 | Asparagine synthetase (ASNS) | 9.7 | 4.8 |
| 243283_at | 54677 | Carnitine O-octanoyltransferase (CROT) | -3.5 | |
| 202402_s_at | 833 | Cysteinyl-tRNA synthetase (CARS) | 4.2 | |
| 231790_at | 29958 | Dimethylglycine dehydrogenase (DMGDH) | 30.4 | |
| Probe set ID | Gene ID1 | Gene title | Fold change | |
|---|---|---|---|---|
| 2-OH-BDE472 | 2-OH-BDE852 | |||
| 213712_at | 54898 | Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 2 (ELOVL2) | -3.0 | |
| 234513_at | 83401 | Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3 (ELOVL3) | -4.4 | |
| 205278_at | 2571 | Glutamate decarboxylase 1 (GAD1) | 4.4 | |
| 202721_s_at | 2673 | Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) | 3.9 | 4.0 |
| 1560013_at | 23042 | Pyridoxal-dependent decarboxylase domain containing 1 (PDXDC1) | 6.2 | |
| 242775_at | 22949 | Leukotriene B4 12-hydroxydehydrogenase (LTB4DH) | -3.9 | |
| 213672_at | 4141 | Methionine-tRNA synthetase (MARS) | 5.4 | 11.7 |
| 225520_at | 25902 | methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L) | 3.2 | 3.6 |
| 206340_at | 9971 | Nuclear receptor subfamily 1, group H, member 4 (NR1H4) | 3.3 | |
| 202847_at | 5106 | Phosphoenolpyruvate carboxykinase 2 (PCK2) | 5.1 | |
| 223062_s_at | 29968 | Phosphoserine aminotransferase 1 (PSAT1) | 3.0 | |
| 214474_at | 5565 | Protein kinase, AMP-activated, beta 2 non-catalytic subunit (PRKAB2) | 3.5 | |
| 214437_s_at | 6472 | Serine hydroxymethyltransferase 2 (SHMT2) | 3.2 | |
| 200802_at | 6301 | Seryl-tRNA synthetase (SARS) | 3.0 | |
| 225779_at | 10999 | Solute carrier family 27 (fatty acid transporter), member 4 (SLC27A4) | -4.2 | -5.4 |
| 200628.s.at | 7453 | Tryptophanyl-tRNA synthetase (WARS) | 4.2 | |
| 212048_s_at | 8565 | Tyrosyl-tRNA synthetase (YARS) | 3.6 | |
1, Gene ID according with Entrez Gene; 2, Cells were incubated with test compounds at 10 AM for 24h as described in Section 2. Significant (P<0.05) changes in mRNA expression levels in treatment group over control (fold changes) are listed. ER stress-responsive genes are highlighted in bold.
system. The excessive accumulation of unfolded proteins into the ER, however, causes ER stress and is associated with several confor- mational diseases. To alleviate ER stress, mammalian cells activate the unfolded protein response (UPR), which inhibits translation to prevent further accumulation of unfolded proteins, and induces the transcription of ER chaperone and ERAD component genes. In some cases, apoptotic cell death is induced to safely dispose of injured cells (Pahl, 1999; Yoshida, 2007).
OH-PBDEs induced key regulators of ER stress, such as a well-known ER chaperone that belongs to the heat shock pro- tein 70 family, HSPA5 (also known as BiP or GRP78), and an ER transmembrane protein that senses the accumulation of unfolded proteins, EIF2AK3/PERK. PERK induces ATF4-mediated dramatic increases in the expression of the pro-apoptotic transcrip- tion factor DDIT3/CHOP and of proteins involved in amino-acid metabolism such as asparagine synthetase and amino acid trans- porters (Ron and Walter, 2007; Yoshida, 2007). In the current study, we observed the modulation of metabolic genes such as gluta- mate decarboxylase 1, methylenetetrahydrofolate dehydrogenase, phosphoenolpyruvate carboxykinase 2 and six aminoacyl tRNA synthetases, all of which were previously shown to be regulated during ER stress in human cells (Murray et al., 2004). In addi- tion to CHOP (14-fold), both OH-PBDEs dramatically increased the expression of a number of stress-responsive, pro-apoptotic and cell cycle arrest genes like protein phosphatase GADD34 (6-10- fold), GADD45a (about 9-fold) and TRIB3 (10-fold), which are also induced during ER stress via the PERK pathway (Ohoka et al., 2005). The induction of these and other genes involved in apoptosis/cell death (e.g. BCL10, PDCD5) was generally more significant for 2-OH- BDE85, which may explain the higher cytotoxicity of this compound (Fig. 1).
2-OH-BDE85 also modulated a greater number of transcription factors and regulators. The highest induction corresponded to a stress response gene, ATF3 (12-18-fold). Also highly up-regulated by both compounds was XBP1 (about 7-fold), a transcription fac- tor that is essential to the function of cells that produce large amounts of secretory proteins and another important mediator of the ER stress response. In agreement with our results, XBP1 is transcriptionally activated during ER stress and it induces the expression of several ER chaperones and ERAD component genes (Ron and Walter, 2007; Yoshida, 2007). This may explain the up- regulation of two members of the heat shock protein family that act
as molecular chaperones to restore correct protein folding during ER stress (DNAJB9, DNAJC3) and a member of the ERAD machinery (HERPUD1) in our study. We have highlighted known ER stress- responsive genes in Table 2 (in bold).
3.5. Validation of gene expression changes
Microarray data were validated by real-time quantitative RT- PCR. We chose seven genes that were up-regulated in the microarray experiment because of their involvement in ER stress response (ATF3, DDIT3, GADD45A, HSPA5, TRIB3) or in response to dioxin (CIP1B1, TIPARP). In addition, we analysed two genes that were down-regulated in the microarray experiment, which are known to play important roles in the normal function of adrenocortical carcinoma cells (ADRA2A, SREBF1). We monitored the expression of these genes in H295R cells incubated with a range of concentrations of 2-OH-BDE47 or 2-OH-BDE85 (0.01, 0.1, 1 and 10 MM). At 10 µM, there was a good agreement between gene regulation in the Affymetrix GeneChip analysis and in the real-time RT-PCR (Fig. 5). Fold changes of expression derived from both techniques were generally similar, with the exception of DDIT, ATF3 and TRIB3, for which the RT-PCR fold change was much higher than that in the microarray experiments. This is likely to be due to the higher sensitivity of the RT-PCR. No expres- sion changes were detected, however, when H295R cells were incubated with lower concentrations of 2-OH-BDE47 or 2-OH- BDE85 (0.01-1 µM) (supplementary data, Fig. 2). Further studies are thus warranted not only to identify the exact mechanism by which OH-PBDEs induce ER stress, but also to determine whether the UPR can be elicited in vivo as a consequence of OH- PBDE bioaccumulation. Estimates based on the accumulation of certain OH-PBDEs, like OH-BDE47, in human serum samples (rang- ing between 0.5 and 6ngg-1 lipid weight; Athanasiadou et al., 2008) indicate that the serum concentration is within the pico- molar range, which is several orders of magnitude lower than the medium concentration of OH-BDEs that elicited toxicity to H295R cells in this study. However, more information is required on the concentration of these compounds in human blood before the possible in vivo relevance of this toxic effect can be accurately determined.
Recently, Van Boxtel et al. (2008) performed Affymetrix microar- ray analysis in embryonic fibroblasts from Zebrafish exposed to
(A)
ATF3
DDIT3
50
TRIB3
Fold Change
GADD45a
40
TIPARP
30
HSPA5
20
CYP1B1
10
0
Control
2-OH- BDE47
2-OH- BDE85
Control
2-OH- BDE47
2-OH- BDE85
RT-PCR
Microarray
(B)
1.6
ADRA2
Fold Change
SREBF1
1.2
0.8
0.4
0
Control
2-OH-
2-OH- BDE85
Control
2-OH- BDE47
2-OH- BDE85
BDE47
RT-PCR
Microarray
another BDE47 metabolite, 6-OH-BDE47. The main result was a mild altered expression of two functional groups of genes involved in proton transport and carbohydrate metabolism, which agreed with their proposed mechanism of toxicity for 6-OH-BDE47 in Zebrafish involving the uncoupling of oxidative phosphorylation. However, it is difficult to compare their results with the current ones, since the authors used a different model organism, a dif- ferent PBDE metabolite and a lower concentration (1 µM) of the agent.
3.6. Summary
In summary, the two OH-PBDE congeners tested in the current study were not toxic to adrenocortical cells at <1 p.M. However, both OH-PBDEs were able to inhibit the proliferation of H295R adrenocortical carcinoma cells in a dose-dependent manner in the micromolar range. This was accompanied by a significant cell cycle arrest at G0/G1. Despite their cytotoxicity, we did not observe the formation of DNA lesions or any evidence of excision repair activity. Instead, our results suggest that ER stress is implicated in the OH-PBDE-mediated adrenocortical toxicity. In particular, OH- PBDEs (10 (M) activate the molecular signature of one of the arms of UPR, namely the PERK pathway. We therefore propose that the bioaccumulation of OH-PBDEs may cause disruption of the adreno- cortical secretory pathways via ER stress. Whilst ER stress explains the cytotoxicity of these compounds in vitro, more work is required to establish whether this could result in impairment of the adreno- cortical gland in vivo.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors thank Ms. Jennifer Higgins and Ms. Rachel Kwok for their assistance with the statistical analysis. This work was supported by the National Natural Scientific Foundation of China [NSFC-40590390 to J.F.] and partly supported by the National Nat- ural Scientific Foundation of China [NSFC-40590393 to J.F.]; the Chinese Academy of Sciences [KZCX2-YW-403 to J.F., O733241001 to R.S.]; and ECNIS (Environmental Cancer Risk, Nutrition and Indi- vidual Susceptibility) a network of excellence operating within the European Union 6th Framework Program, Priority 5: “Food Quality and Safety” [513943 to P.B.F., M.S. & G.D.D.J.]. Comet assay studies in the laboratory of G.D.D.J. were supported by Cancer Research UK [C13560/A46 to G.D.D.J.]; and Fellowships from the Hope Founda- tion [to G.D.D.J.].
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi: 10.1016/j.toxlet.2008.11.011.
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