Comparisons of differential gene expression elicited by TCDD, PCB126, βNF, or ICZ in mouse hepatoma Hepa1c1c7 cells and C57BL/6 mouse liver
Introduction
The aryl hydrocarbon receptor (AhR) is a ligand-dependent basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor activated by structurally diverse synthetic, natural, and dietary compounds including the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Denison and Nagy, 2003, Denison et al., 2011). Although putative endogenous AhR ligands including Kynurenine (Opitz et al., 2011), 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxilic acid methyl ester (ITE) (Henry et al., 2010, Quintana et al., 2010) and formylindolo[3,2-b]carbazole (FICZ) (Wei et al., 1999) have been identified, its true endogenous ligand remains elusive (Denison and Nagy, 2003, Nguyen and Bradfield, 2008).
AhR activation following ligand binding causes dissociation of 90 kDa heat shock protein (HSP90), AhR-interacting protein (AIP; also known as ARA9 or XAP2), and p23, followed by translocation to the nucleus and dimerization with the AhR nuclear translocator (ARNT) (Denison and Nagy, 2003, Denison et al., 2011, Hankinson, 1995). The liganded AhR-ARNT complex then binds to dioxin response elements (DREs; core sequence 5′-GCGTG-3′) eliciting changes in gene expression (Denison and Nagy, 2003, Denison et al., 2011, Dere et al., 2011a, Hankinson, 1995). Recent evidence also suggests AhR-mediated differential gene expression independent of DREs (Beischlag et al., 2008, Denison et al., 2011, Dere et al., 2011b, Huang and Elferink, 2012, Tanos et al., 2012).
TCDD and related compounds elicit species-specific responses including teratogenicity, immunotoxicity, hepatotoxicity, and carcinogenicity (Denison et al., 2011, Hankinson, 1995, Poland and Knutson, 1982) that are mostly, if not entirely, AhR-mediated (Denison and Heath-Pagliuso, 1998, Denison et al., 2011, Gonzalez and Fernandez-Salguero, 1998). For example, AhR null mice are resistant to TCDD-mediated toxicity (Gonzalez and Fernandez-Salguero, 1998). Furthermore, while the potency of AhR ligands is determined by comparisons to TCDD, not all elicit the same effects reported with TCDD (Henry et al., 2010, McKillop and Case, 1991, Murray et al., 2010, Safe et al., 1999, Yin et al., 2012).
Selective modulation of AhR-mediated gene expression (Denison et al., 2011, Jin et al., 2012, Kremoser et al., 2007, Murray et al., 2010, Pansoy et al., 2010, Safe et al., 1999, Yin et al., 2012) is thought to be similar to selective estrogen receptor modulators (SERMs) and selective peroxisome-proliferator activated receptor modulators (SPPARMs) (Berger et al., 2003, Frasor et al., 2004, Sears et al., 2007). Selective modulation involves the induction of a ligand-dependent intermediary conformation that can range from complete inactivity to full activation (Berger et al., 2003, Denison et al., 2011, Kremoser et al., 2007). Different ligand-induced conformations change the surface topology of the activated receptor complex leading to the differential recruitment of co-activators and co-repressors in a gene-, cell-, and tissue-dependent manner that can result in ligand-specific gene expression changes (Berger et al., 2003, Brzozowski et al., 1997, Kremoser et al., 2007, Smith and O’Malley, 2004, Zhang et al., 2008). For example, the SERMs, tamoxifen and raloxifene, reposition helix 12 compared to 17β-estradiol such that they exhibit weaker agonist activity (Brzozowski et al., 1997, Levenson and Jordan, 1999). Selective AhR modulator (SAhRM) development has largely focused on immunosuppression and tumor growth inhibition (Jin et al., 2012, Murray et al., 2010, Safe et al., 1999, Yin et al., 2012). More specifically, alkyl polychlorinated dibenzofurans inhibit mammary tumor growth through an AhR-dependent mechanism absent of Cyp1a1 induction and toxicity (Safe et al., 1999) while 1-allyl-3-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-1H-indazole (SGA360) elicits AhR-mediated immunosuppression independent of DREs without Cyp1a1 induction (Murray et al., 2010). Meanwhile, TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 3,3′4,4′-pentachlorobiphenyl (PCB126) exhibit different ligand-dependent co-activator recruitment to the AhR with varying Cyp1a1 induction efficacy (Zhang et al., 2008), consistent with ligand-dependent co-activator and co-repressor recruitment in a promoter-, cell-, tissue- and species-specific manner indicative of selective modulation.
The ability of various chemicals to selectively modulate the AhR could have important implications for risk assessment as current approaches assume a common mode of action using toxic equivalency factors (van den Berg et al., 2000). In order to examine if AhR ligands which activates the canonical AhR pathway demonstrate selective modulation, differential gene expression elicited TCDD, PCB126, β-naphthoflavone (βNF), and indolo-[3,2b]-carbazole (ICZ) was examined in mouse Hepa1c1c7 cells and C57BL/6 liver samples. Although each ligand exhibits high AhR binding affinity, Cyp1a1 mRNA induction and the induction of aryl hydrocarbon hydroxylase activity (Boobis et al., 1977, Chen et al., 1995, Chen et al., 2010, Denison and Nagy, 2003, Denison et al., 2011, Kopec et al., 2008, Pohjanvirta et al., 2002), they are structurally diverse with different metabolism kinetics. Therefore, global gene expression profiles were compared not only to identify conserved differential expression but also to investigate divergent and ligand-specific gene expression changes suggestive of SAhRM activity.
Section snippets
In vitro treatment
All in vitro studies were performed as previously described (Dere et al., 2006). Briefly, Hepa1c1c7 cells (Dr. O. Hankinson, University of California, Los Angeles, CA) were cultured in phenol-red free DMEM/F12 media (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS; Hyclone, Logan, UT), 2.5 μg/mL amphotericin B (Invitrogen), 50 μg/mL gentamycin (Invitrogen), 100 U/mL penicillin (Invitrogen), and 100 μg/mL streptomycin (Invitrogen). Cells were maintained under standard culture
In vitro microarray analysis
Time-dependent changes elicited by 10 nM TCDD (Dere et al., 2006), 10 μM βNF, 100 nM PCB126, and 1 μM ICZ were evaluated in Hepa1c1c7 hepatoma cells. A total of 288 (130 induced and 158 repressed) TCDD-, 183 (125 induced and 58 repressed) βNF-, 119 (91 induced and 28 repressed) PCB126-, and 131 (78 induced and 53 repressed) ICZ-elicited gene expression changes (|fold-change| ≥ 1.5 and P1(t) ≥ 0.9999) were identified (Fig. 1). Cyp1a1 showed the greatest induction while Alb was most repressed by all
Discussion
In this study, TCDD-, PCB126-, βNF- and ICZ-elicited differential gene expression was compared in Hepa1c1c7 cells and C57BL/6 liver samples. Although these ligands are structurally diverse with different metabolism and elimination pharmacokinetics, all bind the AhR with high affinity (Bohonowych and Denison, 2007) and elicit AhR-mediated differential gene expression. TCDD and PCB126 are metabolized slowly, or not at all, with in vivo elimination rates ranging from days to weeks in rodents (
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Supplementary data
Supplementary Fig. S1 Correlation analysis of differential gene expression between custom cDNA and Agilent oligonucleotide microarrays.
Supplementary Figs. S2 and S3 Rank fold-change correlation of common differentially regulated genes.
Supplementary Table 1 Common and model-specific differentially regulated genes.
Supplementary Tables 2–8 Analyzed microarray datasets including previously published datasets with newly annotated probes.
Acknowledgements
This work was supported by the National Institute of Environmental Health Sciences Superfund Basic Research Program (NIEHS SBRP P42ES04911). The authors would like to thank Dr. Darrell R. Boverhof for his support with in vivo studies and Lyle D. Burgoon for his help with the microarray analyses.
References (64)
- et al.
6-substituted-1,3,8-trichlorodibenzofurans as 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonists in the rat: structure activity relationships
Toxicology
(1989) - et al.
6-Methyl-1,3,8-trichlorodibenzofuran (MCDF) as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6 mice
Toxicology
(1989) - et al.
2,2’,4,4’,5,5’-Hexachlorobiphenyl as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist in C57BL/6J mice
Toxicol. Appl. Pharmacol.
(1989) - et al.
Regulation of CYP1A1 by indolo[3,2-b]carbazole in murine hepatoma cells
J. Biol. Chem.
(1995) - et al.
Immunosuppressive activities of polychlorinated dibenzofuran congeners: quantitative structure-activity relationships and interactive effects
Toxicol. Appl. Pharmacol.
(1988) - et al.
The structure-dependent effects of heptachlorodibenzofuran isomers in male C57BL/6 mice: immunotoxicity and monooxygenase enzyme induction
Fundam. Appl. Toxicol.
(1990) - et al.
Panning for SNuRMs: using cofactor profiling for the rational discovery of selective nuclear receptor modulators
Drug Discovery Today
(2007) - et al.
Selective oestrogen receptor modulation: molecular pharmacology for the millennium
Eur. J. Cancer
(1999) - et al.
Mutagenicity, carcinogenicity and toxicity of beta-naphthoflavone, a potent inducer of P448
Biochem. Pharmacol.
(1991) - et al.
Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis
Biochem. Pharmacol.
(2000)
Comparison of acute toxicities of indolo[3,2-b]carbazole (ICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in TCDD-sensitive rats
Food Chem. Toxicol.
Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on hepatic and uterine estrogen receptor levels in rats
Toxicol. Appl. Pharmacol.
Selective modulation of promoter recruitment and transcriptional activity of PPARgamma
Biochem. Biophys. Res. Commun.
UV-induced CYP1A1 gene expression in human cells is mediated by tryptophan
Chem. Biol. Interact.
2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced porphyria in genetically inbred mice: partial antagonism and mechanistic studies
Toxicol. Appl. Pharmacol.
Ligand-dependent interactions of the Ah receptor with coactivators in a mammalian two-hybrid assay
Toxicol. Appl. Pharmacol.
6-Methyl-1,3,8-trichlorodibenzofuran as a 2,3,7,8-tetrachlorodibenzo-p-dioxin antagonist: inhibition of the induction of rat cytochrome P-450 isozymes and related monooxygenase activities
Mol. Pharmacol.
The aryl hydrocarbon receptor complex and the control of gene expression
Crit. Rev. Eukaryot. Gene Expr.
Distinct properties and advantages of a novel peroxisome proliferator-activated protein [gamma] selective modulator
Mol. Endocrinol.
Persistent binding of ligands to the aryl hydrocarbon receptor
Toxicol. Sci.
Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells
Science
Comparison of beta-naphthoflavone and 3-methylcholanthrene as inducers of hepatic cytochrome(s) P-448 and aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity
Mol. Pharmacol.
Temporal and dose-dependent hepatic gene expression patterns in mice provide new insights into TCDD-Mediated hepatotoxicity
Toxicol. Sci.
Molecular basis of agonism and antagonism in the oestrogen receptor
Nature
Abnormal liver development and resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in mice carrying a mutation in the DNA-binding domain of the aryl hydrocarbon receptor
Toxicol. Sci.
dbZach toxicogenomic information management system
Pharmacogenomics
A mechanism-based mathematical model of aryl hydrocarbon receptor-mediated CYP1A induction in rats using beta-naphthoflavone as a tool compound
Drug Metab. Dispos.
The Ah receptor: a regulator of the biochemical and toxicological actions of structurally diverse chemicals
Bull. Environ. Contam. Toxicol.
Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals
Annu. Rev. Pharmacol. Toxicol.
Exactly the same but different: promiscuity and diversity in the molecular mechanisms of action of the aryl hydrocarbon (dioxin) receptor
Toxicol. Sci.
In vivo-in vitro toxicogenomic comparison of TCDD-elicited gene expression in Hepa1c1c7 mouse hepatoma cells and C57BL/6 hepatic tissue
BMC Genomics
Genome-wide computational analysis of dioxin response element location and distribution in the human, mouse, and rat genomes
Chem. Res. Toxicol.
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2020, Reproductive ToxicologyCitation Excerpt :To our knowledge, this is the first study characterizing human placental trophoblast gene expression following activation of the AhR. Previously, several studies have described genome-wide gene expression profiles in liver in response to AhR agonists e.g. TCDD in human and rat primary hepatocytes [59], mice liver [60] and A549 lung carcinoma cells [61] as well as in benzo[a]pyrene exposed human T lymphocytes [62]. Since AhR is differentially expressed in different tissues and cell types [4–6], it is important to identify tissue and cell type-specific cellular targets in response to AhR ligands.