Tetrabromobisphenol A activates the hepatic interferon pathway in rats

Abstract

Tetrabromobisphenol A (TBBPA) is a widely used flame retardant in printed circuit boards, paper, and textiles. In a two-year study, TBBPA showed evidence of uterine tumors in female Wistar-Han rats and liver and colon tumors in B6C3F1 mice. In order to gain further insight into early gene and pathway changes leading to cancer, we exposed female Wistar Han rats to TBBPA at 0, 25, 250, or 1000 mg/kg (oral gavage in corn oil, 5×/week) for 13 weeks. Because at the end of the TBBPA exposure period, there were no treatment-related effects on body weights, liver or uterus lesions, and liver and uterine organ weights were within 10% of controls, only the high dose animals were analyzed. Analysis of the hepatic and uterine transcriptomes showed TBBPA-induced changes primarily in the liver (1000 mg/kg), with 159 transcripts corresponding to 132 genes differentially expressed compared to controls (FDR = 0.05). Pathway analysis showed activation of interferon (IFN) and metabolic networks. TBBPA induced few molecular changes in the uterus. Activation of the interferon pathway in the liver occurred after 13-weeks of TBBPA exposure, and with longer term TBBPA exposure this may lead to immunomodulatory changes that contribute to carcinogenic processes.

1. Introduction

Tetrabromobisphenol A (TBBPA) is a high production volume brominated flame retardant (Malkoske et al., 2016), used in printed circuit boards, paper, and textiles (U. S. EPA, 2015; Zhou et al., 2014). TBBPA exposure occurs from breast milk (Carignan et al., 2012; Harrad and Abdallah, 2015; Nakao et al., 2015), food ingestion (e.g. fish (Svihlikova et al., 2015)), industrial exposures (Zhou et al., 2014), dust in the home (Di Napoli-Davis and Owens, 2013), and at waste sites (Liu et al., 2016). TBBPA also accumulates in marine life and may be toxic to various fish species (He et al., 2015; Tang et al., 2015).

TBBPA caused clear evidence of uterine adenocarcinomas in female Wistar Han rats [Crl:WI(Han)] in a 2-year study (Dunnick et al., 2015; National Toxicology Program, 2014). These TBBPA-induced uterine tumors were highly malignant with metastases to the liver, pancreas, kidney, thyroid and other organ systems. Other TBBPA carcinogenic findings occurred in the liver, lower intestine, and vascular systems of male mice (National Toxicology Program, 2014). TBBPA has been classified as probably carcinogenic to humans (Group 2a) by the Interagency for Research on Cancer (IARC) based on sufficient evidence for carcinogenicity found in the 2-year rodent studies and mechanistic information reported in the literature (Grosse et al., 2016).

In this study we looked for TBBPA-induced transcriptomic changes in the liver because this organ is a primary site for metabolism of hormones and other chemicals (Tsuchiya et al., 2005), and in the uterus, a target site for TBBPA-induced tumors in rats (National Toxicology Program, 2014). The TBBPA carcinogenic effect in the uterus is of concern because endometrial tumors are a common malignancy in women with an estimated 50,000 new cases per year in the U.S. (Siegel et al., 2013), and one million new cases per year worldwide (Webb, 2015). Uterine cancer is predicted to be one of the three leading cancers in women by 2030 (Rahib et al., 2014). The majority of human uterine tumors are endometrial carcinomas (George et al., 2015); the same type of uterine tumors seen in rats after TBBPA exposure (National Toxicology Program, 2014). Environmental factors are thought to play a role in the development of uterine cancer (Lichtenstein et al., 2000), including chemical and hormone effects (e.g. tamoxifen and estrogen) (IARC, 2012).

TBBPA is a nongenotoxic chemical, and toxicokinetic studies of TBBPA in the female rat did not reveal any specific accumulation of the parent compound or metabolites in the uterus (compared to that in other organ systems) (Knudsen et al., 2014). Thus, these 13-week TBBPA studies were undertaken to identify molecular alterations in the liver and/or uterus to help characterize early changes along the pathway to cancer.

2. Materials and methods

2.1. Experimental design

Tetrabromobisphenol A (CAS No. 79-94-7; Albemarle Corporation (Baton Rouge, LA), lot M032607 K) (Fig. 1) was prepared for oral gavage administration in corn oil to deliver TBBPA at doses of 0, 25, 250, or 1000 mg/kg body weight in a volume of 5 mL/kg body weight. Female Wistar Han IGS rats (Crl:WI(Han)) (25 animals/dose level) were obtained from Charles River (Raleigh, NC). 1000 mg/kg was a dose at which TBBPA induced uterine tumors in the female rat (National Toxicology Program, 2014), and transcriptomic patterns of liver and uterus were examined at this dose level. Two lower doses were added (25 and 250 mg/kg) to examine the uterus for transcriptomic changes, because this was the target organ in the 2-year cancer study. At the start of the study the animals were 5–6 weeks of age. The animals were housed two per cage. Tap water and NTP-2000 diet (Zeigler Brothers, Inc. Gardners, PA) were made available for ad libitum consumption.

Fig. 1.

TBPPA structure.

Body weights were obtained at one day prior to dosing and weekly thereafter. The treatment schedule was five days per week, excluding weekends and holidays, for up to 13 weeks. Animals were treated for a minimum of two consecutive days (within 24 h) prior to necropsy. Animals were not treated on the morning of scheduled sacrifice. At sacrifice, animals were euthanized with CO₂. At necropsy uterine and liver organ weights were taken, and uterine and liver samples fixed in formalin for histopathologic evaluation at all dose levels. Sections were taken from the uterus (control and all dose levels) and liver (control and high dose) and flash frozen for the molecular studies.

The uterus with horns, vagina, and ovaries were harvested. The ovaries and vagina from each animal were fixed in formalin. The uterus was weighed then transected along the midline, including uterine body, such that one half of the uterus (including horn and body) was flash frozen in liquid nitrogen for RNA extraction. The other half of the uterus (including horn and body) was laid out, stretched with minimal tension, and pinned onto a piece of heavy card stock and placed in formalin. This was to prevent curling and shrinkage of the uterine body and horn so that it could be trimmed longitudinally without curling. This allowed appropriate microscopic interpretation of any changes.

The liver was removed and weighed, and then approximately a one-gram sample of the left lobe was collected and minced into approximately 3 mm cubes while on a weigh boat sitting in dry ice. The cubes were then frozen in liquid nitrogen in the weigh boat and, once frozen, transferred to three labeled, 2.0 mL cryotubes and flash frozen in liquid nitrogen. The liver samples were collected and frozen within 5 min of each animal’s sacrifice. An adjacent section (~3 mm) of the remaining left lobe of the liver was collected and placed in a labeled cassette and fixed in formalin for histopathology.

The care of animals on this study was according to NIH procedures as described in the “The U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals”, available from the Office of Laboratory Animal Welfare, National Institutes of Health, Department of Health and Human Services, RKLI, Suite 360, MSC 7982, 6705 Rockledge Drive, Bethesda, MD 20892-7982 or online at http://grants.nih.gov/grants/olaw/olaw.htm#pol. The protocol was approved by the laboratory where the animals were housed and dosed (Alion Science and Technology Animal Care and Use Committee).

2.2. Uterine and liver RNA preparation and microarray hybridization

A section of the frozen liver and one half of the frozen uterus (flash frozen and stored at −80° C) were placed into RNAlater^® (Ambion, Inc., Austin, TX). RNA was extracted from control and treated liver and uterus to identify any molecular changes at a dose that caused uterine tumors (1000 mg/kg). RNA extraction was performed on tissues from 15 to 16 randomly selected animals per group.

RNA was extracted from the flash frozen uterine or liver tissues using the Invitrogen PureLink Mini kit (Invitrogen cat# 12183-018A, Carlsbad, CA) according to the manufacturer’s protocol. Frozen tissue samples were lysed and homogenized in TRIzol reagent (Invitrogen) using a rotor–stator homogenizer. Isolation of RNA was performed according to the mini kit protocol. On-column deoxyribonuclease (DNase) treatment was performed using the Invitrogen PureLink DNase kit (Invitrogen) to purify the RNA samples. RNA concentration and quality were measured on a Bioanalyzer (Agilent Technologies, Santa Clara, CA). Samples were aliquoted and stored at −80°C until they were analyzed.

Total RNA was used to synthesize double-stranded cDNA for each sample using Affymetrix GeneChip^® Expression Analysis with 3’amplication two-cycle target labeling and control reagents (Affymetrix Inc. Santa Clara, CA). The cDNA served as a template to synthesize biotin-labeled antisense cRNA using an in vitro transcription (IVT) labeling kit. Labeled cRNA was fragmented and hybridized to the Affymetrix Rat Genome 230 2.0 Genechip^® Array. Array hybridization, washing, and staining were performed according to the Affymetrix recommended protocol EuKGE_Ws2v5. The chips were scanned using an Affymetrix GeneChip^® Scanner 3000. Quality control measurements were evaluated to determine if the data derived from the arrays were of sufficient quality prior to comparisons for differential expression.

2.3. Microarray data analysis

Gene expression analysis was conducted using Affymetrix Rat Genome 230 2.0 GeneChip^® arrays (Affymetrix, Santa Clara, CA). Total RNA was amplified as directed in the Affymetrix 3′ IVT Plus kit protocol. 15 µg of amplified biotin-aRNAs were fragmented and 12.5 µg were hybridized to each array for 16 h at 45 °C in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Array slides were stained with streptavidin/phycoerythrin utilizing a double-antibody staining procedure and then washed for antibody amplification according to the GeneChip^® Hybridization, Wash and Stain Kit and user manual. Arrays were scanned in an Affymetrix Scanner 3000 and data was obtained using the GeneChip^® Command Console and Expression Console Software (AGCC; Version 3.2 and Expression Console; Version 1.2).

2.4. Data normalization

Probe intensity data from all Rat Genome 230 version 2 Affymetrix GeneChip® arrays were read into the R software environment (http://www.R-project.org) directly from. CEL files using the R/affy package (Gautier et al., 2004). Probe-level data quality was assessed using image reconstruction, histograms of raw signal intensities and hierarchical clustering of samples. Normalization was carried out using the robust multi-array average (RMA) method using all probe intensity data sets together (Irizarry et al., 2003). The RMA method adjusts the background intensities of perfect match (PM) probes, applies quantile normalization, and calculates final expression measures using the Tukey median polish algorithm. RMA scatterplots were used as an additional quality control measure.

2.5. Statistical assessment of differential gene expression

Gene expression between control and treated liver and uterine samples was evaluated for each probe set using a bootstrap t-test approach. Pairwise tests were conducted while controlling the false discovery rate (FDR) at the 5% level. All statistical calculations were performed in the ORIOGEN software package using 10,000 bootstrap samples (Peddada et al., 2005).

Ingenuity Pathway Analysis database (www.Ingenuity.com) was used to compare experimental gene expression signatures to thousands of genomic signatures derived from published microarray data sets. This data mining approach is based on rank-based statistical procedures and is analogous to the Gene Set Enrichment method (Subramanian et al., 2005).

The upstream regulator analysis presented here is based on known relationships between transcriptional factor molecules and targets stored in the Ingenuity Knowledge Base. For each transcription factor, an overlap p-value is used to compare the overlap between the number of known transcription factor targets and the number of targets found in the TBBPA gene list. The activation z-score is used to compare the activation state of each transcription factor as described in the literature with the direction of change in TBBPA expression relative to control samples that are found in the current study. In addition, the interferome database (www.interferome.org) was used to identify significant transcripts in the interferon pathway (Rusinova et al., 2013).

2.6. Nanostring analysis

Microarray results were confirmed using the nCounter platform by NanoString© (www.nanostring.com) utilizing a Custom Code-Set consisting of 10 TBBPA responsive genes that were significantly altered by about 2-fold from control after microarray analysis. Genes evaluated by Nanostring were Agrn, Mx1, Mx2, Irf7, Usp18, Oas1a, Oas1b, Sng, Tsx, and Usp18, using the housekeeping genes Gapdh, Hprt1, Med15 and Rpl7 for normalization. For liver samples in Nanostring analysis, 100 ng of amplified cDNA, obtained utilizing the Nugen™ Ovation Pico WTA System (v2), was used. Gene expression was quantified on the nCounter Digital Analyzer™ and raw and normalized counts were generated with nSolver (v2.5)™ software. All data passed nSolver ‘s QA/QC.

2.7. Infection screening

Sentinel animals were randomly selected for parasite evaluation, gross observation for evidence of disease, and serum collection for serology at 4 weeks after receipt (5 rats) and at the end of the study (5 rats). Sentinels were from the vendor’s same animal room as the experimental rats and were subjected to the same environmental conditions. Serum samples were shipped to an independent laboratory (Idexx Bioresearch, Columbia, MO) for bacterial and viral titer testing. Serum was tested for antibodies to Mycoplasm pulmonis, RPV, RMV, KRV, H1, PMV, RCV/SDAV, RTV, and Sendai virus. All tests were negative.

3. Results

3.1. Body and organ weights and histopathologic findings

There was little or no treatment-related toxicity after 13 weeks of TBBPA exposure using conventional toxicity endpoints. This included no treatment-related effects on survival, body weight, or organ weights (Table 1). There were no treatment-related microscopic lesions in the liver or uterus.

Table 1.

Body weight, liver, and uterus weight in female Wistar Han rats after 13-weeks of TBBPA dosing.

Dose (mg/kg)	Terminal Body wt. (g)	Liver wt. (g)	Relative Liver Weight	Uterus wt (g)	Relative Uterus Wt.	N
0	241.7 ± 3.704	8.13 ± 0.190	33.55 ± 0.563	0.92 ± 0.081	3.87 ± 0.362	22–23
25	230.6 ± 3.435	7.74 ± 0.143	33.60 ± 0.427	1.07 ± 0.077	4.65 ± 0.31	25
250	236.1 ± 4.148	7.89 ± 0.1	33.45 ± 0.411	0.97 ± 0.070	4.14 ± 0.309	24
1000	244.6 ± 3.036	8.71 ± 0.11*	35.63 ± 0.29**	0.97 ± 0.06	3.99 ± 0.286	21

Relative organ weight = organ weight/body weight.

^*p < 0.05.

^**p < 0.01.

3.2. Liver transcriptomic alterations

Using a false discovery rate threshold of 0.05, there were 159 differentially expressed liver transcript probes (Table 2). These 159 transcripts corresponded to expression level changes in 132 genes (6 genes were identified by two probes; Supplement 1). The heat map of these TBBPA liver transcript patterns showed a clear difference between control and TBBPA treated liver (Fig. 2).

Table 2.

TBBPA-induced liver transcripts after 13-weeks of TBBPA dosing (1000 mg/kg).

Fold Change	ID	Symbol	Entrez Gene Name
7.363	1367668_a_at	Scd2	stearoyl-Coenzyme A desaturase 2
3.428	1387283_at	MX18	MX dynamin-like GTPase 1
3.264	1389034_at	USP18*	ubiquitin specific peptidase 18
3.065	1368736_at	Tsx	testis specific X-linked gene
2.773	1371152_a_at	OAS18	2′–5′-oligoadenylate synthetase 1, 40/46 kDa
2.633	1382314_at	*	ISG15 ubiquitin-like modifier
2.258	1371076_at	CYP2B6	cytochrome P450, family 2, subfamily B, polypeptide 6
2.199	1394401_at	ELOVL6	ELOVL fatty acid elongase 6
2.189	1382902_at	HERC6*	HECT and RLD domain containing E3 ubiquitin protein ligase family member 6
1.939	1379748_at	IFI44L*	interferon-induced protein 44-like
1.904	1383564_at	IRF7*	interferon regulatory factor 7
1.844	1388056_at	Oas1b	2′–5′ oligoadenylate synthetase 1B
1.833	1381556_at	Ddx60*	DEAD (Asp-Glu-Ala-Asp) box polypeptide 60
1.827	1369698_at	ABCC3*	ATP-binding cassette, sub-family C (CFTR/MRP), member 3
1.826	1371015_at	Mx1/Mx2*	MX dynamin-like GTPase 1
1.734	1387137_at	COMP	cartilage oligomeric matrix protein
1.703	1377497_at	OASL*	2′–5′-oligoadenylate synthetase-like
1.666	1367707_at	FASN	fatty acid synthase
1.658	1370913_at	RSAD2*	radical S-adenosyl methionine domain containing 2
1.627	1376908_at	IFIT3*	interferon-induced protein with tetratricopeptide repeats 3
1.565	1381960_at	ROPN1L	rhophilin associated tail protein 1-like
1.543	1381206_at	PLCXD2	phosphatidylinositol-specific phospholipase C, X domain containing 2
1.527	1383424_at	CMPK2*	cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial
1.500	1390127_at	DIXDC1	DIX domain containing 1
1.490	1391507_at	ZNF4678	zinc finger protein 467
1.486	1370379_at	PRSS8	protease, serine, 8
1.468	1376920_at	LOC500013	similar to sterile alpha motif domain containing 9-like
1.462	1388164_at	HLA-E	major histocompatibility complex, class I, E
1.459	1391754_at	LOC100910735/Oas1i	2 ' – 5 ' oligoadenylate synthetase 1I
1.455	1379656_a_at	CORO2A	coronin, actin binding protein, 2A
1.451	1373267_at	SH3YL1	SH3 and SYLF domain containing 1
1.433	1383357_a_at	RELL1	RELT-like 1
1.421	1383448_at	IRF98	interferon regulatory factor 9
1.415	1367854_at	ACLY*	ATP citrate lyase
1.408	1381014_at	IFI44*	interferon-induced protein 44
1.407	1367708_a_at	FASN*	fatty acid synthase
1.378	1378581_at	CCDC62	coiled-coil domain containing 62
1.350	1391702_at	ZNF446*	zinc finger protein 446
1.349	1371694_at	DPYSL2	dihydropyrimidinase-like 2
1.346	1369716_s_at	Lgals5	lectin, galactose binding, soluble 5
1.332	1390312_at	LOC684193	similar to sterile alpha motif domain containing 9-like
1.328	1385252_at	TRIM6–TRIM34	TRIM6–TRIM34 readthrough
1.320	1393044_at	CMPK2	cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial
1.314	1380071_at	PARP12*	poly (ADP-ribose) polymerase family, member 12
1.299	1373514_at	RNF213*	ring finger protein 213
1.287	1388152_at	MAP2*	microtubule-associated protein 2
1.269	1388979_at	SMNDC1	survival motor neuron domain containing 1
1.266	1373506_at	FAM102A	family with sequence similarity 102, member A
1.247	1371901_at	DDHD2	DDHD domain containing 2
1.247	1391463_at	Ddx588	DEAD (Asp-Glu-Ala-Asp) box polypeptide 58
1.245	1390641_at	ZNF346	zinc finger protein 346
1.237	1377878_at	FGFBP3	fibroblast growth factor binding protein 3
1.236	1372705_at	CHERP	calcium homeostasis endoplasmic reticulum protein
1.225	1374337_at	RNF2138	ring finger protein 213
1.224	1368835_at	STAT1*	signal transducer and activator of transcription 1, 91 kDa
1.222	1370803_at	ZWINT8	ZW10 interacting kinetochore protein
1.220	1373090_at	SSR1	signal sequence receptor, alpha
1.215	1397764_at	KCTD5	potassium channel tetramerization domain containing 5
1.215	1389883_at	TMEM65	transmembrane protein 65
1.212	1383621_at	MBTPS2	membrane-bound transcription factor peptidase, site 2
1.211	1372930_at	SP110*	SP110 nuclear body protein
1.210	1373288_at	ST5*	suppression of tumorigenicity 5
1.203	1376339_at	WWC3*	WWC family member 3
1.200	1398435_at	SLC22A15*	solute carrier family 22, member 15
1.199	1371996_at	AEBP2	AE binding protein 2
1.198	1372779_at	B3GNT2*	UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2
1.198	1383034_at	Rybp*	RING1 and YY1 binding protein
1.195	1367693_at	YWHAH	tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta
1.194	1395081_at	NCOA7*	nuclear receptor coactivator 7
1.187	1399157_at	URI1	URI1, prefoldin-like chaperone
1.184	1369559_a_at	CD47*	CD47 molecule
1.180	1369978_at	PRPSAP2	phosphoribosyl pyrophosphate synthetase-associated protein 2
1.177	1382540_at	PRPF40A	PRP40 pre-mRNA processing factor 40 homolog A (S. cerevisiae)
1.174	1394761_at	ARHGAP42	Rho GTPase activating protein 42
1.172	1374482_at	CTPS2	CTP synthase 2
1.172	1382177_at	PML*	promyelocytic leukemia
1.169	1383158_at	AGFG1	ArfGAP with FG repeats 1
1.168	1380960_at	CORO2A	coronin, actin binding protein, 2A
1.167	1388997_at	ARF3	ADP-ribosylation factor 3
1.166	1387770_at	Ifi27*	interferon, alpha-inducible protein 27
1.163	1384362_at	ZBTB24	zinc finger and BTB domain containing 24
1.159	1375431_at	C2orf69	chromosome 2 open reading frame 69
1.157	1370244_at	CTSV*	cathepsin V
1.156	1374815_at	STARD3NL	STARD3 N-terminal like
1.155	1389686_at	PRKX	protein kinase, X-linked
1.152	1373563_at	SNRNP27	small nuclear ribonucleoprotein 27 kDa (U4/U6.U5)
1.151	1376492_at	UNKL	unkempt family zinc finger-like
1.150	1392916_at	MAP7	microtubule-associated protein 7
1.148	1370351_at	TDRD78	tudor domain containing 7
1.147	1379669_at	ARHGAP42	Rho GTPase activating protein 42
1.147	1373670_at	STAT2*	signal transducer and activator of transcription 2, 113 kDa
1.146	1389646_at	CDC23	cell division cycle 23
1.142	1388930_at	TMEM123	transmembrane protein 123
1.131	1372360_at	ABI1	abl-interactor 1
1.128	1390387_at	SH3D19	SH3 domain containing 19
1.119	1374637_at	TMCO6	transmembrane and coiled-coil domains 6
1.115	1374707_at	CCAR2	cell cycle and apoptosis regulator 2
1.113	1374406_at	KLHDC2*	kelch domain containing 2
1.112	1384022_at	AGFG1	ArfGAP with FG repeats 1
1.104	1368217_at	RALBP1*	ralA binding protein 1
1.101	1373974_at	OSBP	oxysterol binding protein
1.099	1375639_at	E2F6	E2F transcription factor 6
1.080	1384971_at	DEPDC7	DEP domain containing 7
−1.102	1383244_at	IQCE*	IQ motif containing E
−1.122	1393123_at	C8G	complement component 8, gamma polypeptide
−1.125	1369898_a_at	GIP	gastric inhibitory polypeptide
−1.137	1371916_at	MSRB*	methionine sulfoxide reductase B1
−1.137	1380624_at	VANGL1	VANGL planar cell polarity protein 1
−1.145	1370682_at	LILRA6*	leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 6
−1.146	1376761_at	HDAC4	histone deacetylase 4
−1.152	1370202_at	PLA2G16*	phospholipase A2, group XVI
−1.156	1368399_a_at	CPQ*	carboxypeptidase Q
−1.160	1370853_at	CAMK2N1	calcium/calmodulin-dependent protein kinase II inhibitor 1
−1.160	1370267_at	GSK3B	glycogen synthase kinase 3 beta
−1.168	1376410_at	MMP17	matrix metallopeptidase 17 (membrane-inserted)
−1.170	1398351_at	USP7	ubiquitin specific peptidase 7 (herpes virus-associated)
−1.174	1367586_at	Ldha/RGD1562690	lactate dehydrogenase A
−1.176	1377112_at	CDA*	cytidine deaminase
−1.194	1399070_at	SETD5	SET domain containing 5
−1.199	1372490_at	GPD1L*	glycerol-3-phosphate dehydrogenase 1-like
−1.207	1368190_at	REN	renin
−1.220	1383989_at	SOX4*	SRY (sex determining region Y)-box 4
−1.222	1372195_at	TNNC2*	troponin C type 2 (fast)
−1.245	1396188_at	RHOJ	ras homolog family member J
−1.253	1381193_at	LPGAT1*	lysophosphatidylglycerol acyltransferase 1
−1.253	1374048_at	NRTN	neurturin
−1.263	1370375_at	GLS2	glutaminase 2 (liver, mitochondrial)
−1.292	1393949_at	HYAL3	hyaluronoglucosaminidase 3
−1.293	1382981_at	AHI1	Abelson helper integration site 1
−1.317	1374073_at	SLC46A1	solute carrier family 46 (folate transporter), member 1
−1.349	1387307_at	HAL*	histidine ammonia-lyase
−1.445	1397552_at	EML4	echinoderm microtubule associated protein like 4
−1.451	1387656_at	SLC4A1	solute carrier family 4 (anion exchanger), member 1 (Diego blood group)
−1.503	1393971_at	LOC102549203	uncharacterized LOC102549203
−1.658	1393910_at	Fam13a*	family with sequence similarity 13, member A
−1.736	1387052_at	GPT	glutamic-pyruvate transaminase (alanine aminotransferase)
−1.801	1368520_at	APOA4	apolipoprotein A-IV
−1.809	1387391_at	CDKN1A*	cyclin-dependent kinase inhibitor 1A (p21, Cip1)

^*Interferon pathway transcript.

Fig. 2.

TBBPA (1000 mg/kg) liver transcript heat map.

Nanostring analysis of selected transcripts confirmed the direction of the TBBPA-induced transcript change (Fig. 3), although Agrn and Sncg were minimally responsive to TBBPA by Nanostring analysis. Selected liver transcript signals were plotted by individual animals to display variance of control and TBBPA groups. Generally, transcript responses showed the same elevated direction of response of TBBPA treatment group compared to control (Fig. 4).

Fig. 3.

Selected Liver TBBPA (1000 mg/kg) transcripts confirmed by Nanostring analysis.

Fig. 4.

Plots of selected TBBPA (1000 mg/kg) induced-liver transcripts. The gene expression value shown refers to the RMA normalized gene expression measure described in the methods.

Ingenuity Pathway enrichment analysis provided evidence that the interferon (IFN) pathway was the most significant pathway affected by TBBPA treatment (Table 3). This led us to explore the IFN pathway more closely. There were 59 significantly altered TBBPA hepatic transcripts in the IFN pathway (www.interferome.org). Transcripts altered by TBBPA that are known to play critical roles in the interferon pathway included Irl-7, Mx1, Oas1, Isfg15, Ddx60, Stat1 and Stat2. Also upregulated were TBBPA transcripts involved in liver xenobiotic metabolism, including genes regulating fatty acid metabolism (Scd2, Cyp2b6, Elovl6, Herc6, Fasn) (Table 2).

Table 3.

Analysis of significant TBBPA liver transcripts and pathways (1000 mg/kg)^*.

Ingenuity Canonical Pathways	−log(p-value)	Ratio	z-score	Molecules
Interferon Signaling	8.85E00	1.94E-01	2.449	OAS1,IRF9,STAT1,IFIT3,STAT2,ISG15,MX1
Activation of IRF by Cytosolic Pattern Recognition Receptors	4.47E00	7.94E-02	1.342	IRF9,IRF7,STAT1,STAT2,ISG15
Cell Cycle: G1/S Checkpoint Regulation	3.27E00	6.25E-02	−1.000	E2F6,GSK3B,CDKN1A,HDAC4
Cyclins and Cell Cycle Regulation	2.94E00	5.13E-02	NaN	E2F6,GSK3B,CDKN1A,HDAC4
Pancreatic Adenocarcinoma Signaling	2.46E00	3.77E-02	NaN	E2F6,RALBP1,STAT1,CDKN1A
Acetyl-CoA Biosynthesis III (from Citrate)	2.23E00	1E00	NaN	ACLY
Role of JAK1, JAK2 and TYK2 in Interferon Signaling	2.06E00	8.33E-02	NaN	STAT1,STAT2
Estrogen-mediated S-phase Entry	2.06E00	8.33E-02	NaN	E2F6,CDKN1A
JAK/Stat Signaling	2.06E00	4.17E-02	NaN	STAT1,STAT2,CDKN1A

^*Analysis using ingenuity.com.

There were few TBBPA-induced changes in the uterine transcriptome. Using a false discovery rate threshold of 0.05, there were 4 differentially expressed transcripts at 1000 mg/kg, 8 at 250 mg/kg, and 5 at 25 mg/kg. All 17 transcripts at various dose levels were less than 1.5 fold changed versus control uterine transcript expression levels. None of the 17 uterine transcripts were common among the three TBBPA exposure levels examined (25, 250, and 1000 mg/kg), and none overlapped the 138 TBBPA liver transcripts found to be altered (1000 mg/kg). These transcript changes were not considered to alter organ function.

4. Discussion

In this study we found that TBBPA induced molecular changes in the liver after 13-weeks of exposure (1000 mg/kg), while there were few altered transcripts in the uterus. TBBPA caused liver expression changes in 159 significant Affymetrix probes (relative to controls [FDR ≤ 0.05]) which mapped to 132 genes. The TBBPA hepatic transcriptome included transcripts with functions in the IFN pathway (Noureddin et al., 2015; Sathish and Yuan, 2011; Schmeisser et al., 2010; Schneider et al., 2014) and in xenobiotic and fatty acid metabolism.

The TBBPA hepatic transcripts included upregulation of Scd2 (steraroly-coenzyme A desaturase 2), Elovl-6 (fatty acid elongase 6), and FasN (fatty acid synthase). Scd2 is a crucial enzyme in the synthesis of monounsaturated fatty acids which are required for maintaining a normal epidermal permeability barrier function, and are components of triglycerides (Miyazaki et al., 2005). Alterations in Scd2 levels may affect lipid content in non-hepatic organs (de Moura et al., 2016) where level and type of lipid is critical to function. Elovl-6 is one of six mammalian enzymes responsible for fatty acid elongation beyond 16 carbons to produce very long chain fatty acids. The FASN enzyme catalyzes de novo synthesis of fatty acids (Dorn et al., 2010). TBBPA also increased levels of the Cyp2b6, a transcript induced by phenobarbital and in xenobiotic metabolism (Liu et al., 2015). The expression of Cyp2b6 can have a 20–250 fold inter-individual variation (Wang and Tompkins, 2008). Because liver is a major site for chemical exposure (Hakk et al., 2000; Knudsen et al., 2014; Kuester et al., 2007) and is involved in estradiol metabolism (Gosavi et al., 2013; Raftogianis et al., 2000), these TBBPA-induced liver changes could affect hormone levels.

TBBPA induced IFN pathway transcripts (www.interferome.org) previously identified in liver cells (Rusinova et al., 2013). This included transcripts associated with IFN pathway regulation (e.g. Stat1, Stat2, Ilf7, Irf9, Pml) (Khodarev et al., 2012); antiviral activity (e.g. Mx1, Mx2, Ifit3, Isg15); and regulation of immune response (DDX58, Oas1) (Hertzog et al., 2011). Some of the TBBPA IFN pathway transcripts (e.g. Isg15) may be involved in hepatic cancer (Li et al., 2014), and could also play a role in the liver cancer that occurred in mice in the 2-year TBBPA study (National Toxicology Program, 2014). Ifl27 (induced by TBBPA) may promote cell proliferation (Hsieh et al., 2015), and is associated with tumorigenesis and invasion (Li et al., 2015).

Like TBBPA, tamoxifen causes uterine tumors (IARC, 2012), and induces interferon pathway transcripts (e.g. MX1 and interferon regulatory factors) in model systems (Dabydeen et al., 2015; Fawzy et al., 2012; Schild-Hay et al., 2009). Exposure of myeloid cells to a related chemical, bisphenol A, also stimulated interferon signaling (Panchanathan et al., 2015). These TBBPA findings are supported by recent in vitro studies which showed TBBPA alterations in IFN production in human cells (Almughamsi and Whalen, 2016).

While TBBPA has little activity as an estrogen receptor agonist or antagonist (Hamers et al., 2006), a feedback loop between estrogen signaling and IFN signaling has been reported (Panchanathan et al., 2015, 2010). Upregulation of interferon pathways by TBBPA and tamoxifen could affect estrogen signaling and ultimately the development of uterine cancer. The ability of TBBPA to interact with sulfotransferase (Gosavi et al., 2013) could disrupt estrogen homeostasis (Sanders et al., 2016). In addition, TBBPA can cause oxidative damage and disruption of thyroid hormone signaling (He et al., 2016; Iakovleva et al., 2016). Further work is needed to determine how these various TBBPA effects work together to cause cancer and how age affects biologic outcome (Hines, 2008).

Decreases in immune function can facilitate cancer development (Yang and Rosenberg, 2016), and in several studies TBBPA was found to suppress the immune system. TBBPA was an immunosuppressant in in vitro cell systems including suppression of NK cell activity (Hurd and Whalen, 2011; Kibakaya et al., 2009). Mice treated with TBBPA were less able to mount a defense against viral infection (Watanabe et al., 2010). Interferon signaling modulates immune activity, and Irf7 when upregulated in the liver, as occurred in these TBBPA studies (Fig. 5), can regulate the immune system (Wang et al., 2013). Persistent IFN signaling can disrupt immune responsiveness potentially leading to immune suppression (Teijaro, 2016). This TBBPA immunosuppressent activity was thought to contribute to its carcinogenic properties in a recent review of TBBPA studies (Grosse et al., 2016).

Fig. 5.

Proposed Mechanism for TBBPA activation of interferon pathway.

One interpretation of the TBBPA-mediated IFN signature reported in our study might be as a general stress response to repeated chemical exposure. The expression of ‘IFN stimulated genes’ or ‘interferon signaling network genes’ (ISGs) in response to various viral, bacterial and chemical stimuli has been well characterized and described (de Veer et al., 2001; Schneider et al., 2014). In our studies animals were screened for a broad spectrum of infections and none were found, suggesting that the IFN pathway activation was not due to microbial or viral exposure. In addition, liver histopathology showed no overt signs of cell death or organ damage. Exactly, how TBBPA-mediated expression of ISGs in liver might influence or contribute to uterine carcinogenesis is unclear at this time. Although blood cytokine measurements were not performed in this study, it is possible that TBBPA could alter circulating or organ-localized production of interferon to affect ISGs leading to the IFN signature observed here in liver. For example, microrarray analysis in an independent study showed that acute pentachorophenol exposure in C57BL/6 mice increased expression of many interferon responsive transcripts in liver, including Stat1, Stat2, Irf7, Oas, Ifit as well as other IFN regulated genes (Kanno et al., 2013). These authors proposed a scheme where pentachorophenol produced metabolites and oxygenated radicals leading to DNA and protein damage that resulted in pathway activation of the Nrf2 Tir receptor and PRR (pattern response recognition) systems to initiate interferon signaling and expression of ISGs. Another study found in vitro TBBPA exposure disrupted IFN-γ secretion from various human immune cell preparations (Almughamsi and Whalen, 2016). Whether the uterus would respond similarly to TBBPA or other chemical exposures and engage IFN-dependent pathways is an untested hypothesis. IFN polymorphisms are associated with increased risk of cervical cancer (Sun et al., 2015) but the relationship of IFN-related mechanisms with uterine carcinogenesis requires further investigation.

In summary, conventional biochemical and toxicological measures and histologic lesions were not observed in either liver or uterus in female Wistar Han rats after 13 weeks of repeated TBBPA exposure from 25 to 1000 mg/kg. Toxicogenomic analysis showed no substantial changes in uterus but did reveal a robust gene expression in liver that involved activation of the interferon pathway. We speculate that long-term TBBPA exposures could lead to direct or indirect immunomodulatory changes that contribute to carcinogenic processes in the uterus.

HIGHLIGHTS.

• Tetrabromobisphenol A (TBBPA) is a widely used flame retardant.

• Oral administration of TBBPA to rats cause transcriptomic changes in the liver.

• These transcriptomic changes indicated activation of the interferon pathway.