Screening a mouse liver gene expression compendium identifies modulators of the aryl hydrocarbon receptor (AhR)

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dioxin-like compounds (DLC) as well as some drugs and endogenous tryptophan metabolites. Short-term activation of AhR can lead to hepatocellular steatosis, and chronic activation can lead to liver cancer in mice and rats. Analytical approaches were developed to identify biosets in a genomic database in which AhR activity was altered. A set of 63 genes was identified (the AhR gene expression biomarker) that was dependent on AhR for regulation after exposure to TCDD or benzo[a]pyrene and includes the known AhR targets Cyp1a1 and Cyp1b1. A fold-change rank-based test (Running Fisher's test; p-value ≤10⁻⁴) was used to evaluate the similarity between the AhR biomarker and a test set of 37 and 41 biosets positive or negative, respectively for AhR activation. The test resulted in a balanced accuracy of 95%. The rank-based test was used to identify factors that activate or suppress AhR in an annotated mouse liver/mouse primary hepatocyte gene expression database of ∼1850 comparisons. In addition to the expected activation of AhR by TCDD and DLC, AhR was activated by AP20189 and phenformin. AhR was suppressed by phenobarbital and 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) in a constitutive activated receptor (CAR)-dependent manner and pregnenolone-16α-carbonitrile in a pregnane X receptor (PXR)-dependent manner. Inactivation of individual genes in nullizygous models led to AhR activation (Pxr, Ghrhr, Taf10) or suppression (Ahr, Ilst6st, Hnf1a). This study describes a novel screening strategy for identifying factors in mouse liver that perturb AhR in a gene expression compendium.

Introduction

Adverse outcome pathways (AOPs) are defined as a series of mechanistically-linked key events starting with a molecular initiating event (MIE) in which a chemical or other stressor interacts with a target proceeding to an adverse outcome in a tissue (Ankley et al., 2010, Pery et al., 2013). A group of AOPs that converge on adverse effects in the liver involve a group of ligand-activated transcription factors including the aryl hydrocarbon receptor (AhR). AhR is a member of the basic region helix-loop-helix/period-aryl hydrocarbon nuclear translocator-simple-minded (bHLH/PAS) family of transcription factors that controls a variety of developmental and physiological events including the induction of drug and xenobiotic metabolizing enzymes, response to hypoxia, and hormone receptor function (Bock and Kohle, 2009, McMillan and Bradfield, 2007). For humans, rats and mice the mechanism for AhR activation has similar features. In its nonligand-bound form, AhR is primarily found in the cytoplasm complexed with a number of proteins including the 90-kDa heat shock protein (Hsp90) (Heid et al., 2000). Upon ligand binding, the AhR complex undergoes a conformational change resulting in complex dissociation, translocation to the nucleus, and subsequent AhR heterodimerization with the aryl hydrocarbon nuclear translocator (ARNT), another member of the bHLH/PAS family (Bock and Kohle, 2009, McMillan and Bradfield, 2007). The AhR/ARNT complex, in cooperation with co-regulator proteins, interact with enhancer sequences in the promoters of target genes called dioxin-responsive elements (DREs), and either stimulate or suppress target gene transcription (Hankinson, 1995, Schmidt and Bradfield, 1996). Several genes involved in phase I (e.g., CYP1A1, CYP1A2, CYP1B1) and phase II (e.g., NQO1 and ALDH4) xenobiotic metabolism contain functional DRE sequences in their promoter regions and are transcriptionally activated by a variety of AhR agonists (Schmidt and Bradfield, 1996).

AhR is activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; also generically referred to as dioxin), one of the most potent known AhR agonists, as well as dioxin-like chemicals (DLCs) that include other polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs). Compounds which activate AhR include many other industrial compounds and byproducts, widely used pharmaceuticals, and several classes of chemoprotective phytochemicals such as the flavonoids, indole-3-carbinol, and related compounds (reviewed in Denison et al., 2011). In general, the ligands share similar structural features, i.e., small, aromatic, and planar. However, the ligands can differ markedly in how they affect AhR function (Safe et al., 2013).

Initial interest in the mechanistic basis for AhR action arose from the relationship between activation of this receptor, carcinogenicity in laboratory animals, and concerns over occupational and environmental exposures to TCDD and DLCs (Knerr and Schrenk, 2006). Chronic exposure to TCDD leads to increased incidence of hepatocellular adenoma, cholangiocarcinoma and cholangioma, predominantly in female rats. In mice, both males and females develop liver tumors following TCDD treatment (Budinsky et al., 2013). Lifetime cancer bioassays in rodents have been carried out for other dioxins, furans, and dioxin-like PCBs including 2,3,4,7,8-pentachlorodibenzofuran, a mixture of hexachlorodibenzo-p-dioxins, and PCB126 and PCB118. These studies are notable for a close relationship between liver histopathology, tumor development and the unusual extent and presentation of the hepatic lesions (Goodman and Sauer, 1992, Hailey et al., 2005). The AOP from chronic TCDD or DLC exposure to liver cancer includes chronic activation of AhR, alteration of genes that control cell fate, inhibition of intrafocal apoptosis, increased cell proliferation of previously initiated foci and proliferation of oval cells (Budinsky et al., 2013). Although not considered hepatocarcinogenic themselves, endogenous ligands of AhR (e.g., kynurenine, kynurenic acid and indoles) may modulate the activation of AhR and thus the hepatocarcinogenic process (Budinski et al., 2013). Short-term liver toxicity is not observed in either Ahr-null mice (Gonzalez and Fernandez-Salguero, 1998) or TCDD-resistant Han Wistar/Kupio rats at doses of TCDD that are hepatotoxic in sensitive strains such as Sprague-Dawley rats (Sand et al., 2010). Recent studies with an AHR-null rat model demonstrated that AhR was required for TCDD to induce liver weights and liver xenobiotic metabolism genes (Harrill et al., 2013). Congenic mice homozygous for either the Ahr^b1 or Ahr^d alleles (encoding an AhR with high or low binding affinity for TCDD, respectively) were used to demonstrate that hepatocellular tumor promotion in response to TCDD was dependent on the status of the Ahr locus (Kennedy et al., 2014). Overall, these studies support AhR activation as the MIE in an AOP involving TCDD- or DLC-induced liver cancer.

Increasing evidence points to a role for AhR in fatty liver disease. In humans exposed to dioxin, there is an increased incidence of fatty liver (Lee et al., 2006). Transgenic or pharmacological activation of AhR in mice led to spontaneous fatty liver disease (Lu et al., 2011a, Lu et al., 2011b) by transcriptionally activating the fatty acid translocase FAT/Cd36 gene and increasing fatty acid uptake (He et al., 2011). Moreover, TCDD increases the expression of intestinal and hepatic lipid transport genes including Slc27a, Fabp, Ldlr, and Apob (Angrish et al., 2012). A mouse strain (B6.D2 strain) encoding a low-affinity AhR was resistant to the effects of a high fat western-style diet including increases in body mass, gonadal fat pad mass, liver to body weight ratios, liver damage and steatosis compared to the parent C57BL/6 (B6 strain), which naturally bears the high-affinity AhR (Kerley-Hamilton et al., 2012). Activation of AhR also increased oxidative stress (Lu et al., 2011a, Dalton et al., 2002), a risk factor for nonalcoholic steatohepatitus (NASH), possibly through decreases in superoxide dismutase activity (Sod2) controlled by the mitochondrial NAD-dependent deacetylase sirtuin 3 (Sirt3) (He et al., 2013). TCDD treatment led to decreased expression of the hepatic triglyceride hydrolase carboxylesterase 3 (Ces3) in an AhR-dependent manner in mouse liver and forced expression of Ces3 ameliorated steatohepatitus caused by a methionine and choline deficient diet (Matsubara et al., 2012). In summary, an hypothesized AOP leading to steatohepatitis in mice involves (1) activation of AhR (the MIE); (2) increased expression of fatty acid uptake genes (Cd36, Slc27a, Fabp, Ldlr, Apob) and decreased Ces3 expression involved in triglyceride degradation; (3) increased accumulation of triglycerides in hepatocytes (“first hit”); (4) increases in oxidative stress through decreases in Sirt3-controlled Sod2 (“second hit”); and (5) steatohepatitis.

The ability to predict AhR activation or suppression in gene expression profiles would be useful to identify chemicals and other factors that modulate AhR. In the present study, a gene expression biomarker that predicts AhR activation or suppression in mice was characterized by identifying genes consistently altered by AhR activation in wild-type but not AhR-null mice. This biomarker coupled with a rank-based statistical test was found to accurately predict activation of AhR and was used to screen a mouse liver gene expression compendium of ∼1850 comparisons to find chemicals and other factors that modulate AhR activity. The predictions could be used as a starting point to link AhR activation and the adverse outcomes of liver cancer and steatosis.