Elsevier

Toxicology Letters

Volume 223, Issue 1, 23 October 2013, Pages 52-59
Toxicology Letters

Comparisons of differential gene expression elicited by TCDD, PCB126, βNF, or ICZ in mouse hepatoma Hepa1c1c7 cells and C57BL/6 mouse liver

https://doi.org/10.1016/j.toxlet.2013.08.013Get rights and content

Highlights

  • Gene expression elicited by AhR ligands TCDD, PCB126, βNF and ICZ was compared.

  • AhR ligands TCDD, PCB126, βNF and ICZ regulate a common subset of genes.

  • AhR ligand elicited gene expression diverges as duration of exposure increases.

  • TCDD, PCB126, βNF and ICZ selectively modulate the aryl hydrocarbon receptor.

Abstract

The aryl hydrocarbon receptor (AhR) is a promiscuous receptor activated by structurally diverse synthetic and natural compounds. AhR activation may lead to ligand-specific changes in gene expression despite similarities in mode of action. Therefore, differential gene expression elicited by four structurally diverse, high affinity AhR ligands (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 nM, 30 μg/kg), 3,3′,4,4′,5-pentachlorobiphenyl (PCB126; 100 nM, 300 μg/kg), β-naphthoflavone (βNF; 10 μM, 90 mg/kg), and indolo[3,2-b]carbazole (ICZ; 1 μM)) in mouse Hepa1c1c7 hepatoma cells and C57BL/6 mouse liver samples were compared. A total of 288, 183, 119, and 131 Hepa1c1c7 genes were differentially expressed (|fold-change|  1.5, P1(t)  0.9999) by TCDD, βNF, PCB126, and ICZ, respectively. Only ∼35% were differentially expressed by all 4 ligands in Hepa1c1c7 cells. In vivo, 661, 479, and 265 hepatic genes were differentially expressed following treatment with TCDD, βNF, and PCB126, respectively. Similar to Hepa1c1c7 cells, ≤34% of gene expression changes were common across all ligands. Principal components analysis identified time-dependent gene expression divergence. Comparisons of ligand-elicited expression between Hepa1c1c7 cells and mouse liver identified only 11 common gene expression changes across all ligands. Although metabolism may explain some ligand-specific gene expression changes, PCB126, βNF, and ICZ also elicited divergent expression compared to TCDD, suggestive of selective AhR modulation.

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.

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