Hepatocyte-Specific Deletion of TIPARP, a Negative Regulator of the Aryl Hydrocarbon Receptor, Is Sufficient to Increase Sensitivity to Dioxin-Induced Wasting Syndrome

Abstract

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of dioxin (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin; TCDD), which includes thymic atrophy, steatohepatitis, and a lethal wasting syndrome in laboratory rodents. Although the mechanisms of dioxin toxicity remain unknown, AHR signaling in hepatocytes is necessary for dioxin-induced liver toxicity. We previously reported that loss of TCDD-inducible poly(adenosine diphosphate [ADP]-ribose) polymerase (TIPARP/PARP7/ARTD14), an AHR target gene and mono-ADP-ribosyltransferase, increases the sensitivity of mice to dioxin-induced toxicities. To test the hypothesis that TIPARP is a negative regulator of AHR signaling in hepatocytes, we generated Tiparp^fl/fl mice in which exon 3 of Tiparp is flanked by loxP sites, followed by Cre-lox technology to create hepatocyte-specific (Tiparp^fl/flCre^Alb) and whole-body (Tiparp^fl/flCre^CMV; Tiparp^Ex3−/−) Tiparp null mice. Tiparp^fl/flCre^Alb and Tiparp^Ex3−/− mice given a single injection of 10 μg/kg dioxin did not survive beyond days 7 and 9, respectively, while all Tiparp^+/+ mice survived the 30-day treatment. Dioxin-exposed Tiparp^fl/flCre^Alb and Tiparp^Ex3−/− mice had increased steatohepatitis and hepatotoxicity as indicated by greater staining of neutral lipids and serum alanine aminotransferase activity than similarly treated wild-type mice. Tiparp^fl/flCre^Alb and Tiparp^Ex3−/− mice exhibited augmented AHR signaling, denoted by increased dioxin-induced gene expression. Metabolomic studies revealed alterations in lipid and amino acid metabolism in liver extracts from Tiparp^fl/flCre^Alb mice compared with wild-type mice. Taken together, these data illustrate that TIPARP is an important negative regulator of AHR activity, and that its specific loss in hepatocytes is sufficient to increase sensitivity to dioxin-induced steatohepatitis and lethality.

2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin) is a highly toxic environmental contaminant produced during waste incineration and other high-temperature industrial processes, and it remains a global health concern. The toxic effects of dioxin are mediated through its binding to and activation of the aryl hydrocarbon receptor (AHR), which is a member of the basic helix-loop-helix Period-AHR nuclear translocator (ARNT)-Single-minded family of transcription factors. In the canonical AHR signaling pathway, ligand binding to cytosolic AHR causes its translocation to the nucleus where it dimerizes with ARNT (also known as hypoxia-inducible factor 1β). The AHR: ARNT heterodimer then binds DNA sequence elements (termed AHREs or DREs) located within the regulatory regions of its target genes, which include cytochrome P4501A1 (CYP1A1), CYP1B1, and TCDD-inducible adenosine diphosphate (ADP)-ribose polymerase (TIPARP) (Ma et al., 2001; Whitlock, 1999; Zhang et al., 1998). In addition to its roles in dioxin toxicity, xenobiotic metabolism and vascular development (Stevens et al., 2009), AHR has important roles in T-cell differentiation, in the defense against bacterial infections and in gut homeostasis (Moura-Alves et al., 2014; Quintana et al., 2008). To date, >400 AHR ligands have been identified, including dietary compounds (3, 3'-diindolylmethane), various endogenous ligands (kynurenine, 6-formylindolo(3, 2b)carbazole), and environmental contaminants such as dioxin (Denison and Nagy, 2003).

Dioxin causes diverse toxic effects in laboratory rodents including immunosuppression, steatohepatitis, impaired reproduction, and a lethal wasting syndrome (Birnbaum, 1994, 1995; Poland and Knutson, 1982). A single dose of dioxin induces a lethal starvation-like syndrome, which includes decreased gluconeogenesis, liver damage, steatohepatitis and dyslipidaemia, ultimately leading to lethality (Linden et al., 2010; Seefeld et al., 1984). Acute lethality varies widely both among species and between rodent strains. For example, the median lethal dose (LD₅₀) for guinea pigs is 1–2 μg/kg dioxin, whereas the LD₅₀ for hamsters is >5000 μg/kg (Pohjanvirta and Tuomisto, 1994; Poland and Knutson, 1982). In most strains of mice, lethality occurs only 2–3 weeks after a single dose of 115–300 μg/kg of dioxin (Pohjanvirta and Tuomisto, 1994; Poland and Knutson, 1982). There is a roughly 10-fold difference in susceptibility between the high sensitivity C57BL/6 (Ahr^b1 allele) and low sensitivity DBA/2 (Ahr^d allele) mouse strains, which is due to polymorphic variations in their ligand-binding domains (Poland et al., 1994). The molecular mechanisms of dioxin-induced wasting syndrome remain obscure, but transgenic mice overexpressing AHR show increased sensitivity to dioxin toxicities, whereas Ahr^−/− null and Ahr^dbd mice, which express a mutant AHR that does not bind to AHREs, are resistant to the effects of dioxin (Fernandez-Salguero et al., 1996; Lee et al., 2010; Walisser et al., 2005).

TIPARP (PARP7/ARTD14) is an AHR-regulated gene and a member of the poly-adenosine diphosphate (ADP)-ribose polymerase (PARP) family. PARPs are nicotinamide adenine dinucleotide (NAD⁺)-dependent enzymes that use NAD⁺ as a substrate to transfer 1 molecule of ADP-ribose, referred to as mono-ADP-ribosylation (MARylation), or several ADP-ribose moieties, referred to as poly-ADP-ribosylation (PARylation), to specific amino acid residues on themselves and on target proteins (Hottiger et al., 2010). Mono- and poly-ADP-ribosylation are reversible posttranslational modifications involved in several biological processes, such as immune cell function, regulation of transcription, protein expression and DNA repair (Kraus and Hottiger, 2013). We previously reported that TIPARP is a mono-ADP-ribosyltransferase that functions as part of a negative feedback loop to repress AHR activity through a mechanism that involves reduced ligand-induced AHR protein levels and that requires TIPARP’s catalytic activity (MacPherson et al., 2013). Moreover, Tiparp^−/− mice exhibit increased AHR activity but also increased sensitivity to dioxin-induced toxicities, including steatohepatitis, hepatotoxicity and lethal wasting syndrome (Ahmed et al., 2015). Although the mechanisms of dioxin-induced toxicity remain incompletely understood, AHR expression in hepatocytes is needed to generate the adaptive as well as toxic response to dioxin exposure (Walisser et al., 2005).

Here, we describe the generation of Tiparp conditional mutant mice in which exon 3 of the Tiparp gene is flanked by loxP sites, and the subsequent creation of both hepatocyte-specific (Tiparp^fl/flCre^Alb) and whole-body (Tiparp^fl/flCre^CMV; Tiparp^Ex3−/−) TIPARP knockout mice. These mice were used to further investigate the role of TIPARP in AHR signaling and dioxin-induced toxicity.

MATERIALS AND METHODS

Generation of conditional Tiparp null mice

Tiparp^fl/fl mice, where exon 3 of Tiparp was flanked by loxP sites, were generated from embryonic stem (ES) cells purchased from European Conditional Mouse Mutagenesis (EUCOMM; Tiparp^{tm1a(EUCOMM)Wtsi}). ES cells were expanded by the Toronto Centre for Phenogenomics (TCP). Correct targeted recombination was confirmed in 2 of 5 ES cell clones purchased. Briefly, SphI-digested genomic DNA was used in Southern blotting to produce 8.9 kb (wild-type [WT]) and 12.3 kb (tm1a) fragments spanning the 5’ region of the targeted sequence of Tiparp, and in the targeted allele, including the neomycin-resistance (Neo) cassette. The Neo probe was used to reveal additional or random integrations of the targeting vector in the genomes (fragments of incorrect sizes); these ES clones were excluded. PCR primers used to generate the respective probes are provided in Supplementary Table 1. Based on the positive Southern blot results, ES cell clones D3 and G3 were chosen for aggregation and implantation into pseudopregnant surrogate females (performed by TCP). Chimeric Tiparp^+/tm1a mice were bred to C57BL/6 albino females and pups were genotyped to identify those with germline transmission of the conditional mutant Tiparp allele (Tiparp^+/tm1a); only ES clone G3 resulted in successful germline transmission. Some Tiparp^+/tm1a mice were bred to B6.C-Tg(CMV-cre)1Cgn/J (Jackson Labs, Bar Harbor, Maine) to remove the Neo cassette and the targeted exon and leave the lacZ reporter gene (conversion to tm1b allele) and then outbred once to C57BL/6N to remove Tg-(CMV-cre). Mice heterozygous for the tm1b allele were then intercrossed to make homozygous tm1b mice (Tiparp^tm1b/tm1b, referred to as Tiparp^Ex3−/−). Other Tiparp^+/tm1a mice were bred to B6(C3)-Tg(Pgk1-FLPo)10Sykr/J (Jackson Labs) to remove the lacZ and Neo cassettes and leave the targeted exon flanked by LoxP sites (conversion to tm1c allele) and then outbred once to C57BL/6N to remove Tg-(Pgk1-FLPo) (Tiparp^+/tm1c). Tiparp^+/tm1c mice were bred to B6.C-Tg(CMV-cre)1Cgn/J to remove the targeted exon (conversion to tm1d allele) and then outbred once to C57BL/6N to remove Tg-(CMV-cre). Mice heterozygous for the tm1d allele were then intercrossed to make homozygous tm1d mice (Tiparp^tm1d/tm1d; referred to as Tiparp^Ex3−/−). Tiparp^+/tm1c mice were also bred to B6N.Cg-Tg(Alb-cre)21Mgn/J (Jackson Labs) to create hepatocyte-specific Tiparp knock-out mice (Tiparp^tm1c/tm1cCre^Alb; referred to as Tiparp^fl/flCre^Alb. This colony was maintained such that Tiparp^fl/fl female mice were paired with Tiparp^fl/flCre^Alb male mice. Genotypes of all mice were determined by PCR analysis of tail biopsies using PCR primers shown in Supplementary Table 1. The specificity of excision events in Tiparp^fl/flCre^Alb mice was determined by quantitative real time PCR (qPCR) using primer pairs specific for exon 3 of Tiparp compared with primer pairs specific for intron 1 (Supplementary Table 1). Genotype controls used in experiments for the Tiparp^fl/flCre^Alb line are Tiparp^fl/fl (observed to be phenotypically equivalent to WT mice), and for the Tiparp^Ex3−/− are Tiparp^+/+. Both tm1b and tm1d lines were used.

In vivo dioxin treatment studies

All experiments used 8- to 10-week-old male mice. For the acute 6 h exposure studies, Tiparp^Ex3−/− or Tiparp^fl/flCre^Alb mice and their respective strain-specific WT control mice were treated with a single intraperitoneal (i.p.) injection of 100 μg/kg dioxin, and livers were excised and flash frozen 6 h later. For the subacute dioxin toxicity studies, mice were treated with a single i.p. injection of 10 or 100 μg/kg of dioxin and sacrificed on day 6. The 10 μg/kg dose of dioxin was dissolved in a mixture of corn oil and dimethyl sulfoxide (CO:DMSO; 90:10, referred to as CO), while the 100 μg/kg dose of dioxin was dissolved in pure DMSO. For the survival studies, mice were followed for up to 30 days after a single injection of 10 or 100 μg/kg of dioxin. Control mice received equivalent weight-adjusted volumes of CO or DMSO. The time point for euthanization was determined based on endpoint criteria for our study: a loss of 20% body weight or indications of acute distress. All control mice were euthanized to match the endpoints of dioxin-sensitive mice. For food intake studies, mice were housed individually and provided intact pellets of food that were weighed daily; a baseline was determined for each mouse by monitoring for 1 week prior to treatment as described previously (Ahmed et al., 2015). Whole blood was obtained from the saphenous vein for serum alanine aminotransferase (ALT) analysis as described previously in Ahmed et al. (2015). Hepatic glycogen levels were determined from 10 mg of frozen liver using the Glycogen Assay Kit II (Abcam). Liver, thymus, white and brown adipose tissue (WAT and BAT) were dissected and weighed. Livers from Tiparp^Ex3−/− or Tiparp^fl/flCre^Alb mice treated with vehicle, 10 or 100 µg/kg dioxin were collected either on day 6 or on the day of euthanization in the survival studies. Care and treatment of animals followed the guidelines set by the Canadian Council on Animal Care, and all protocols were approved by the University of Toronto Animal Care Committee.

Hepatocytes

Tiparp^fl/flCre^Alb or Tiparp^fl/fl male mice (8- to 10-weeks old) were used to isolate primary hepatocytes. Mouse liver was perfused with liver perfusion medium (Invitrogen) for 10 min followed by liver digestion medium for 10 min. Freshly prepared hepatocytes were seeded at a final density of 0.5 × 10⁶ cells/well onto type I collagen coated 6-well plates in attachment medium (William’s E media, 10% dextran-coated charcoal (DCC) stripped fetal bovine serum (FBS), 1× penicillin/streptomycin, and 10-nM insulin). The medium was changed 2 h after plating, and all experiments were performed on the second day. Ligands were added to the cells in M199 media with 5% DCC-FBS and cells were harvested 16 h after ligand treatment for RNA extraction.

RNA extraction and gene expression analysis

Livers were removed, washed in ice-cold PBS, weighed, and flash frozen in liquid nitrogen. Frozen livers were homogenized in TRIZOL reagent (Life Technologies) and total RNA was isolated using the Aurum RNA isolation kits (BioRad) and reverse transcribed as previously described (Ahmed et al., 2015). Primers used to amplify target transcripts are provided in Supplementary Table 1 or described elsewhere (Ahmed et al., 2015). All genes were normalized to TATA-binding protein levels and analyzed using the comparative C_T (ΔΔ C_T) method.

Chromatin immunoprecipitation (ChIP) assays

ChIP assays were performed as previously described in Lo et al. (2011). Briefly, approximately 100 mg of frozen mouse liver was homogenized in 1% formaldehyde in PBS and incubated for 10 min at room temperature. The homogenate was centrifuged at 8000 × g for 5 min at 4°C. Pellet was washed in ice-cold PBS, centrifuged, and resuspended in 900 μl of TSEI (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate) + 1× Protease Inhibitor Cocktail (Sigma, St Louis, Missouri). Samples were sonicated 10 times for 30 s ON/30 s OFF on the high setting using a Bioruptor (Diagenode). The supernatants were transferred to new microcentrifuge tubes and incubated with rabbit IgG (5 μg; Sigma) and antiAHR (5 μg; SA-210, Enzo) overnight at 4°C under gentle agitation. ChIP samples were washed, the DNA and the ChIP-qPCR was performed as previously described in Lo et al. (2011).

Histology

Hematoxylin and eosin and Oil-Red-O/Hematoxylin staining were performed following standard methods with representative images provided. Paraformaldehyde-fixed or optimal cutting temperature (OCT) compound-embedded tissues were provided to the Histology Core Facility at the Princess Margaret Cancer Centre, Toronto, Ontario, for all histology sample processing, staining and scanning of stained slides.

Western blotting

For hepatic AHR protein detection, whole cell extracts were prepared by homogenizing 100 mg of liver tissue in RIPA lysis buffer. Ten micrograms of total protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were incubated with antiAHR antibody (SA-210) and stripped and then incubated with antiβ-actin antibody (Sigma A-2228).

Metabolomics

Tiparp^fl/fl and Tiparp^fl/flCre^Alb mice were treated with a single intraperitoneal injection of CO: DMSO or 10 μg/kg dioxin. Liver tissue (100 mg) was collected on day 3 and flash frozen in liquid nitrogen. The frozen tissue was extracted and metabolomic analyses were performed by Metabolon (Durham, North Carolina). Raw data received from Metabolon were preprocessed to input missing values and normalized using logarithmic transformation. Shapiro’s test was used to test for normality and Levene’s test was used to test for the homogeneity of variances. Analysis of variance (ANOVA) was used to analyze the differences in group means, followed by Tukey’s-HSD for post hoc correction. The FDR method was used to adjust the p-values for multiple ANOVA tests (one for each metabolite). At an FDR of 10%, all the group comparisons that exhibited a statistically significant difference (post hoc corrected p-values < .05) were considered significant. All the analyses were performed in R version 3.4.1.

Statistical analysis

All data were presented as means and SEM. Two-way ANOVA followed by Sidak’s post hoc test or 1-way ANOVA followed by Tukey’s post hoc test were used to assess statistical significance (p < .05) using GraphPad Prism 6 Software (San Diego, California) or R version 3.4.1.

RESULTS

Generation of Conditional Tiparp^fl/fl Mice

Mice harboring the conditional Tiparp^fl allele were generated from Tiparp^{tm1a(EUCOMM)Wtsi} ES cells purchased from EUCOMM. A partial map of both the Tiparp WT and Tiparp tm1a alleles is shown in Figure 1A. Southern blotting of genomic DNA isolated from 4 different ES cell clones confirmed the correct integration of the tm1a allele in the ES cell clones D3 and G3 (Figure 1B). Mice generated in this study were from ES cell clone G3. Mice harboring the conditional Tiparp^fl allele were generated from the Tiparp tm1a allele by excision of the Neo and LacZ genes through crosses with B6(C3)-Tg(Pgk1-FLPo)10Sykr/J mice, leaving the targeted exon flanked by LoxP sites. Mice heterozygous for the tm1c allele were then intercrossed to make homozygous tm1c mice (Tiparp^fl/fl; tm1c allele).

Figure 1.

Generation of the conditional Tiparp^fl/fl mice. A, Schematic diagram of Tiparp WT allele and the allele with successful recombination of the tm1 targeting construct (Tiparp tm1a allele) and corresponding Southern blot data. Exon numbers reflect known coding exons; white arrowheads represent FRT sites; gray arrowheads represent LoxP sites; LacZ represents lacZ reporter gene; N represents the Neo resistance cassette; S, SphI site; dashed lines indicate the fragment of DNA generated by SphI digestion that was detected with the radiolabeled probes (5’ probe and Neo probe); letters indicate genotyping primers. B, Southern blots show Tiparp allele fragments detected with the radiolabeled probes: ES clones D3 and G3 show correct homologous recombination of the targeting construct (12.3 kb fragment detected with 5’ probe) without additional random integrations (only the 12.3 kb band detected with Neo probe). C, Schematic diagram of Tiparp tm1 alleles following excision of sequence by CRE or FLP recombinases. D, PCR data from mouse tail biopsies. The Tiparp tm1a allele was converted to tm1b using CRE recombinase to remove the Neo cassette and exon 3 located between the LoxP sites. Primer pair D-E amplify a product spanning the excision sites to show that the floxed sequences were removed (420 bp). The Tiparp tm1a allele was converted to tm1c using FLP recombinase to remove the LacZ and Neo cassettes located between the FRT sites. Primer pair A-B amplifies a product spanning the excision site to show that the sequence was flipped out (750 bp). The Tiparp tm1c allele was converted to tm1d using CRE recombinase to remove the floxed exon 3. Primer pair A-E amplify a product spanning the floxed region to show that the exon is removed (310 bp). Sample genotypes: + indicates the Tiparp WT allele; letters indicate the Tiparp tm1 corresponding allele (tm1a, tm1b, tm1c, and tm1d).

Complete and Hepatocyte-Specific Excision of the Tiparp^fl/fl Allele

Using genetrap targeted TIPARP null mice in which a LacZ gene is inserted in front of exon 1 in the Tiparp gene (Schmahl et al., 2007), we reported that loss of TIPARP expression increased the sensitivity of mice to dioxin-induced steatohepatitis and lethality (Ahmed et al., 2015). These findings supported the notion that TIPARP protects against dioxin-induced toxicity by negatively regulating AHR action. Because the type of gene knockout targeting strategy can impact the phenotypes observed from targeting the same gene, we created a complete TIPARP knockout by removal of exon 3 of Tiparp (Tiparp^Ex3−/−) as described in Materials and Methods section. To generate a complete TIPARP null mouse in which exon 3 is removed (Tiparp^Ex3−/−) we used 2 different strategies. In the first approach, mice heterozygous for the tm1c allele were bred to B6.C-Tg(CMV-cre)1Cgn/J mice to remove the targeted exon (Tiparp^Ex3−/−; tm1d allele). In second approach, mice carrying the tm1a allele were bred to B6.C-Tg(CMV-cre)1Cgn/J mice to remove the Neo cassette and the targeted exon and leave the lacZ reporter (Tiparp^Ex3−/−; tm1b allele). A map of the Tiparp tm1c, tm1d, and tm1b alleles is shown in Figure 1C. The correct genotype was confirmed by PCR analysis of genomic DNA (Figure 1D). Tiparp^Ex3−/− mice represent a distinct TIPARP null strain compared with Tiparp targeted knockout using a genetrap approach (Ahmed et al., 2015; Schmahl et al., 2007). To determine whether the loss of TIPARP in hepatocytes is sufficient to increase sensitivity to dioxin-dependent toxicity, we generated mice in which Tiparp was deleted in hepatocytes, Tiparp^fl/flCre^Alb (Figure 2A). Tiparp^fl/flCre^Alb mice were created by breeding mice homozygous for the tm1c allele (Tiparp^fl/fl) with B6N.Cg-Tg(Alb-cre)21Mgn/J mice to remove the targeted exon specifically in hepatocytes (Walisser et al., 2005). For the Tiparp^fl/flCre^Alb line, WT mice were referred to as Tiparp^fl/fl, whereas for the Tiparp^Ex3−/− line, WT mice were referred to as Tiparp^+/+ for simplicity.

Figure 2.

Specificity of Cre^Alb-mediated excision of the Tiparp^fl allele. A, Specificity of Tiparp^fl excision by Cre^Alb was determined by quantitative real-time PCR-based genotyping for excised and unexcised alleles of Tiparp^fl in genomic DNA isolated from various tissues and hepatocytes obtained from Tiparp^fl/fl and Tiparp^fl/flCre^Alb mice. *p < .05 compared with tissue matched Tiparp^fl/fl mice. B, Tiparp^fl excision by Cre^CMV was determined from genomic DNA isolated from livers of Tiparp^+/+ and Tiparp^Ex3−/− mice. Abbreviations: Hep, hepatocytes; WAT, white adipose tissue. C, Increased AHR regulated Cyp1a1, Cyp1a2, Cyp1b1, and Tiparp mRNA levels in expression in hepatocytes isolated from Tiparp^fl/fl and Tiparp^fl/flCre^Alb mice treated with 10-nM dioxin for 6 h. RNA and qPCR were performed as described in the materials and methods section. *p < .05 compared with genotyped match control treated, #p < .05 compared with dioxin-treated Tiparp^fl/fl (n = 3).

To examine the specificity of excision events in Tiparp^fl/flCre^Alb mice, we analyzed various tissues by determining the relative ratios of Tiparp exon 3 compared with intron 1 using qPCR. Tiparp exon 3 was efficiently excised in liver tissue and in isolated hepatocytes (Hepa) in the presence of Cre^Alb, but not in the other tissues examined (Figure 2A). Tiparp exon 3 was not detected in liver tissue isolated from Tiparp^Ex3−/− mice (Figure 2B). In the absence of Cre^Alb, the Tiparp^fl/fl mice showed only the Tiparp^fl-unexcised allele in all tissues and isolated hepatocytes examined (Figure 2A).

Cloning and DNA sequencing revealed that the conditional inactivation of the Tiparp^fl allele accomplished by Cre-mediated deletion of exon 3 (169 bp) results in splicing and fusion between exon 2 (961 bp) and exon 4 (161 bp), leading to the insertion of a premature stop codon. This truncated version of TIPARP contains 311 amino acids, of which the last 4 are the result of a frameshift (the full-length protein is 657 a.a.), and it lacks its tryptophan-tryptophan-glutamate (WWE) and catalytic domains (MacPherson et al., 2013). In agreement with a previous study, cloning and transient transfection of the truncated TIPARP protein failed to inhibit AHR-dependent and dioxin-induced CYP1A1 reporter gene activity (Supplementary Figure 1).

Consistent with TIPARP’s role as a negative regulator of AHR activity, exposure of hepatocytes from Tiparp^fl/flCre^Alb mice for 6 h to 10 nM dioxin increased mRNA expression levels of the AHR target genes Cyp1a1, Cyp1a2 and Cyp1b1 compared with similarly treated hepatocytes from Tiparp^fl/fl mice (Figure 2C). Tiparp mRNA expression levels were markedly decreased in hepatocytes isolated from Tiparp^fl/flCre^Alb mice compared with Tiparp^fl/fl mice. We next examined the effect of TIPARP loss on AHR target gene expression in liver and lung tissues isolated from Tiparp^Ex3−/−, Tiparp^fl/flCre^Alb, and WT mice 6 h after treatment with 100 μg/kg dioxin. Higher Cyp1a1 and Cyp1b1 mRNA levels were observed in both liver and lung from Tiparp^Ex3−/− mice than in Tiparp^Ex3+/+ mice (Figs. 3A and 3B). Tiparp mRNA levels were not detected in liver or lung from Tiparp^Ex3−/− mice compared with Tiparp^+/+ mice (Figure 3C). Significantly increased dioxin-induced Cyp1a1 and Cyp1b1 mRNA levels above those observed in Tiparp^fl/fl mice were only observed in liver and lung from Tiparp^fl/flCre^Alb mice (Figs. 3D and 3E). As expected, TIPARP expression levels were reduced in liver, but not in lung of Tiparp^fl/flCre^Alb compared with Tiparp^fl/fl mice (Figure 3F).

Figure 3.

AHR regulated transcript levels in liver and lung tissue isolate from control and dioxin-treated Tiparp^Ex3−/−, Tiparp^fl/flCre^Alb, and their respective WT mice. Mice were treated with a single injection of 100 μg/kg dioxin in DMSO, or DMSO vehicle alone, and euthanized 6 h later. RNA and qPCR were performed as described in the Materials and Methods section. *p < .05 compared with genotype matched control-treated; #p < .05 compared with dioxin-treated Tiparp^+/+ (A–C) or Tiparp^fl/fl (D–F) (n = 4).

Tiparp^Ex3−/− Mice Exhibit Increased Sensitivity to Dioxin-Induced Toxicity and Lethality

To determine the sensitivity of Tiparp^Ex3−/− mice to dioxin-induced toxicity, these mice and their respective WT controls were injected with a single dose of 10 or 100 μg/kg dioxin and monitored for up to 30 days, as previously described (Ahmed et al., 2015). All Tiparp^Ex3+/+ mice were normal in physical appearance at the end of the 30-day observation period (Figure 4A), while no dioxin-treated Tiparp^Ex3−/− mice (tm1d or tm1b) survived the 30-day experiment (Figure 4A). Tiparp^Ex3−/− mice treated with 100 μg/kg dioxin became weakened and moribund and were humanely euthanized between days 2 and 3, while those treated with 10 μg/kg dioxin were euthanized at day 7. Tiparp^Ex3−/− mice treated with 10 μg/kg dioxin had lost significant body weight by 5 days after treatment (Figure 4B); however, no decrease in food intake was observed (Figure 4C). No changes in food intake or body weight were seen in dioxin-exposed Tiparp^+/+ mice. Serum ALT activity, a marker of hepatotoxicity, was significantly increased in dioxin-treated Tiparp^Ex3−/− mice, while no increase above controls was observed in Tiparp^+/+ mice (Figure 4D). Increased liver weight was observed in dioxin-treated Tiparp^+/+ mice but not in Tiparp^Ex3−/− mice (Figure 4E). Thymic involution, an endpoint associated with dioxin toxicity, responded as expected in both genotypes at day 7 (Figure 4F). A significant decrease in epididymal WAT weight was seen in Tiparp^Ex3−/− mice but not in WT mice (Figure 4G). No differences in BAT weight were observed (data not shown). Hepatic glycogen stores were lower in dioxin-treated Tiparp^Ex3−/− mice than in WT mice (Figure 4H). These data support the importance of TIPARP in regulating AHR action and show that loss of its expression in mice increases their sensitivity to dioxin toxicity.

Figure 4.

Loss of TIPARP increases dioxin-induced hepatotoxicity and lethal wasting syndrome in male mice. A, Kaplan-Meier survival curves for male Tiparp^+/+and Tiparp^Ex3−/− mice treated with a single 10 or 100 µg/kg i.p. injection of dioxin and monitored for 30 days. B, Body weight, (C) food intake, (D) serum ALT activity, (E) liver, (F) thymus, and (G) WAT weight expressed as percentage of total body weight, and (H) hepatic glycogen levels were measured from Tiparp^+/+ and Tiparp^Ex3−/− mice treated with 10 μg/kg dioxin. Data shown are the mean ± SEM (n = 4–5). For (B–G) *p < .05, 2-way ANOVA followed by Tukey’s post hoc test compared with genotype-matched control treated mice. For (H) *p < .05, Student’s t test.

Hepatocyte-Specific Loss of TIPARP Results in Increased Sensitivity to Dioxin-Induced Toxicity and Lethality

Because AHR expression in hepatocytes is required for dioxin-induced liver toxicity, we hypothesized that the loss of TIPARP expression in hepatocytes would enhance dioxin-dependent liver toxicity. To test this hypothesis, we treated Tiparp^fl/flCre^Alb and Tiparp^fl/fl mice with a single i.p. injection of 10 or 100 μg/kg dioxin and monitored the mice for up to 30 days. As expected, all Tiparp^fl/fl mice were normal in physical appearance at the end of the 30-day observation period (Figure 5A). No dioxin-treated Tiparp^fl/flCre^Alb mice survived the 30-day experiment (Figure 5A). Tiparp^fl/flCre^Alb mice treated with 100 μg/kg dioxin appeared weakened and moribund and were humanely euthanized between days 3 and 5, while those treated with 10 μg/kg dioxin were humanely euthanized at day 9. Sensitivity to dioxin-induced lethality was significantly different between Tiparp^fl/flCre^Alb and Tiparp^Ex3−/− mice at both 10 and 100 μg/kg dioxin, suggesting that cells other than hepatocytes also contribute to dioxin lethality in these models. Tiparp^fl/flCre^Alb mice treated with 10 μg/kg dioxin had lost significant body weight by 6 days after treatment (Figure 5B), while no decrease in food intake was observed (Figure 5C). No change in food intake or body weight was seen in Tiparp^fl/fl mice. Serum ALT was significantly increased in dioxin-treated Tiparp^fl/flCre^Albmice at days 3 and 6, but no increase was observed in Tiparp^fl/fl mice (Figure 5D). Increased liver weight was observed in dioxin-treated Tiparp^fl/fl mice but not in Tiparp^fl/flCre^Alb mice (Figure 5E). Decreased thymus weight was seen in both genotypes at day 9 (Figure 5F). Consistent with dioxin-treated Tiparp^Ex3−/− mice, Tiparp^fl/flCre^Alb mice had significantly decreased epididymal WAT levels compared with WT mice (Figure 5G). No difference in BAT weight was observed (data not shown). Hepatic glycogen stores were also decreased in dioxin-treated Tiparp^fl/flCre^Alb mice compared with Tiparp^fl/fl mice (Figure 5H). These findings show that hepatocyte-specific deletion of TIPARP is sufficient to increase dioxin-induced toxicity and lethality.

Figure 5.

Hepatocyte-specific loss of TIPARP increases dioxin-induced hepatotoxicity and lethal wasting syndrome in male mice. A, Kaplan-Meier survival curves for male Tiparp^fl/fland Tiparp^fl/flCre^Alb mice treated with a single 10 or 100 µg/kg i.p. injection of dioxin and monitored for 30 days. B, Body weight, (C) food intake, (D) serum ALT activity, (E) liver, (F) thymus, (G) WAT weight expressed as percentage of total body weight, and (H) hepatic glycogen levels were measured from Tiparp^fl/fl and Tiparp^fl/flCre^Alb mice treated with 10 μg/kg dioxin. Data shown are the mean ± SEM (n = 4–5). For (B–G) *p < .05, 2-way ANOVA followed by Tukey’s post hoc test compared with genotype-matched control treated mice. For (H) *p < .05, Student’s t test.

As an independent measure of liver toxicity, livers were sectioned and stained with hematoxylin and eosin. Vehicle-treated Tiparp^fl/fl and Tiparp^fl/flCre^Alb mice had histologically normal liver architecture (Figure 6A). On day 9, dioxin-treated Tiparp^fl/fl livers exhibited slight hepatocyte cytoplasmic clearing within periportal regions and inflammatory cell infiltration (Figure 6A). In contrast, day 9 Tiparp^fl/flCre^Alb livers were characterized by inflammatory infiltration and a predominant microvesicular steatosis. Six days after dioxin treatment, livers from Tiparp^fl/fl mice displayed distinct inflammatory cell infiltration and increased clearing of the cytoplasm with the appearance of large vacuoles within hepatocytes. Similar findings were observed in livers isolated from dioxin-treated Tiparp^+/+ and Tiparp^Ex3−/− mice (Supplementary Figure 2A).

Figure 6.

Increased liver inflammation and cytokine levels in dioxin-treated Tiparp^fl/flCre^Alb compared with Tiparp^fl/fl mice. A, Representative H&E staining of livers from Tiparp^fl/fl and Tiparp^fl/flCre^Alb mice (n = 4). Control animals were injected with CO and were euthanized on day 9. The asterisks (*) indicate focal inflammatory infiltration, and the arrowheads indicate microvesicular steatosis. All images are to the same scale. Hepatic (B) Il6, (C) Serpine 1, (D) Cxcl2, and (E) F4/80 mRNA levels were determined as described in the methods. Data represent the mean ± SEM (n = 4). *p < .05 2-way ANOVA compared with genotyped-matched control treated mice. ^#P < 0.05 2-way ANOVA compared with dioxin-treated Tiparp^fl/fl mice.

We next determined the mRNA levels of AHR-regulated cytokines and the macrophage marker F4/80 (Casado et al., 2011; Matsubara et al., 2012). Hepatic interleukin 6 (Il6) levels were unaffected by dioxin treatment in both genotypes (Figure 6B). However, dioxin-treated Tiparp^fl/flCre^Alb mice had increased hepatic expression of Serpine 1 (also known as plasminogen activator inhibitor-1; PAI-1), chemokine (C-X-C motif) ligand 2 (Cxcl2) and F4/80 when compared with Tiparp^fl/fl mice (Figs. 6C–E). Similar findings were also seen in livers isolated from dioxin-treated Tiparp^+/+ and Tiparp^Ex3−/− mice (Supplementary Figs. 2B–E). The increased cytokine and F4/80 levels indicated increased hepatic inflammation in dioxin-treated Tiparp^fl/flCre^Alb compared with WT mice.

Hepatocyte-Specific Loss of TIPARP Increases Dioxin-Induced Steatohepatitis

Livers from vehicle-treated Tiparp^fl/fl and Tiparp^fl/flCre^Alb mice were macroscopically normal (Figure 7A). Livers from Tiparp^fl/fl mice were enlarged but only slightly pale in color 9 days after dioxin treatment (Figure 7A). Livers from dioxin-treated Tiparp^fl/flCre^Alb mice were markedly pale in color at 9 days, suggesting a high level of lipid accumulation. We tested for the presence of neutral lipids by Oil-Red-O staining (Figure 7B). Livers from vehicle-exposed Tiparp^fl/fl and Tiparp^fl/flCre^Alb mice were negative for Oil-Red-O staining. On day 9 after dioxin treatment, small droplets of lipid were seen in the livers of Tiparp^fl/fl mice, while those from similarly treated Tiparp^fl/flCre^Alb mice had substantial intracytoplasmic lipid accumulation. Comparable findings were observed in livers isolated from dioxin-treated Tiparp^+/+ and Tiparp^Ex3−/− mice (Supplementary Figs. 3A and 3B).

Figure 7.

Dioxin-induced steatosis is increased in Tiparp^fl/flCre^Alb mice. (A) Livers from male Tiparp^fl/fl and Tiparp^fl/flCre^Alb mice given a single i.p. injection of CO or 10 µg/kg dioxin and euthanized after 9 days (n = 5). B, Oil-Red-O and hematoxylin stained liver sections from Tiparp^fl/fl and Tiparp^fl/flCre^Alb mice. All images are to the same scale. Hepatic mRNA levels of Cd36 (C), Scd1 (D), Srebp1 (E), Ppara (F), and Cyp7a1 (G) were determined as described in the methods. Data represent the mean ± SEM (n = 4). For all data, p < .05 was determined by 2-way ANOVA followed by Tukey’s post hoc test comparison. Significantly different compared with genotype-matched *DMSO- or ^#dioxin-treated Tiparp^fl/fl mice.

We then analyzed the hepatic levels of transcripts encoding genes involved in lipid uptake, lipogenesis and cholesterol/bile acid metabolism. Consistent with previous studies (Lee et al., 2010; Lu et al., 2011), the lipid uptake transporter, scavenger receptor encoded by cluster of differentiation 36 (Cd36), was increased 3-fold by dioxin treatment in Tiparp^fl/fl mice and to a greater extent (8-fold) in similarly treated Tiparp^fl/flCre^Alb mice (Figure 7C). Hepatic expression of lipogenic genes including sterol regulatory element-binding transcription factor 1 (Srebp1), and stearoyl-CoA desaturase (Scd1) were significantly decreased in dioxin treated Tiparp^fl/flCre^Alb mice compared with Tiparp^fl/fl mice (Figs. 7D and 7E). Peroxisome proliferator activating receptor α (Ppara) and Cyp7a1, the rate limiting enzyme bile acid synthesis, were also significantly decreased in Tiparp^fl/flCre^Alb mice compared with Tiparp^fl/fl mice (Figs. 7F and 7G). Similar findings were seen in livers isolated from dioxin-treated Tiparp^+/+ and Tiparp^Ex3−/− mice (Supplementary Figs. 3C–G). These data suggest that the increased sensitivity of Tiparp^fl/flCre^Alb and Tiparp^Ex3−/− mice to dioxin-induced steatohepatitis is due to increased lipid uptake rather than increased hepatic lipogenesis.

Increased AHR Regulated Gene Expression in Tiparp^fl/flCre^Alb Mice After Treatment With 10 μg/kg Dioxin

To identify AHR genes and/or changes in metabolites that might provide insight into the molecular mechanisms regulating the increased dioxin sensitivity of Tiparp^fl/flCre^Alb mice, we analyzed changes in dioxin-induced hepatic mRNA and metabolite levels 3 days after dioxin treatment. Day 3 was chosen because we observed significant increases in serum ALT activity in Tiparp^fl/flCre^Alb mice, and we reasoned that this time point could be used to identify early changes in AHR target gene expression and/or metabolite levels prior to more severe toxicities that ultimately led to death. Tiparp^fl/flCre^Alb mice treated with 10 μg/kg dioxin exhibited increased mRNA expression levels of many AHR target genes including Cyp1a1, Cyp1a2, Ahrr, Nqo1, Nfe2l2 and Serpine1 compared with similarly treated Tiparp^fl/fl mice (Figs. 8A and 8C–H). Cyp1a2 expression was slightly increased in Tiparp^fl/flCre^Alb compared with Tiparp^fl/fl mice, but this difference was not statistically significant (Figure 8B). No significant increase in AHR recruitment to Cyp1a1 was observed (Figure 8I). Significantly higher levels of AHR were recruited to Cyp1b1 in liver extracts from Tiparp^fl/flCre^Alb mice compared with Tiparp^fl/fl mice (Figure 8J). Consistent with our previous study, we observed reduced dioxin-induced proteolytic degradation of total AHR protein in liver extracts from Tiparp^fl/flCre^Alb mice compared with Tiparp^fl/fl mice (Figure 8K).

Figure 8.

Hepatocyte-specific loss of TIPARP increases dioxin-dependent regulation of hepatic AHR target gene expression. Hepatic mRNA levels of Cyp1a1 (A), Cyp1a2 (B), Cyp1b1 (C), Tiparp (D), Ahrr (E), Nqo1 (F) Nfe2l2 (G) Serpine 1 (H) were determined after 3 days of exposure to CO or 10 μg/kg dioxin as described in experimental procedures (n = 4). Recruitment of AHR to Cyp1a1 (I) and Cyp1b1 (J). Data represent the mean ± SEM. Representative AHR (K), and β-actin protein levels were detected by Western blotting after 3 days of treatment. AHR proteins levels were normalized to β-actin levels, n = 4) (L). For all data, p < .05 was determined by 2-way ANOVA followed by Tukey’s post hoc test comparison. Significantly different compared with genotype-matched *DMSO- or ^#dioxin-treated Tiparp^fl/fl mice.

We next did comparative metabolomic analyses on liver extracts from Tiparp^fl/flCre^Alb and Tiparp^fl/fl mice. Principal component analysis (PCA) revealed significant treatment-based separations among the samples, with clear distinctions between the vehicle- and dioxin-treated animals in each genotype. Separations were also apparent between the dioxin-treated Tiparp^fl/fl and Tiparp^fl/flCre^Alb animals (Figure 9A). Of the total of 679 named metabolites examined, 213 were significantly altered (P < 0.05) by dioxin treatment of Tiparp^fl/flCre^Alb mice compared with 124 in similarly treated Tiparp^fl/fl mice (Table 1; Supplementary Tables 2 and 3). Of the 124 metabolites, 74 overlapped with those identified in dioxin-treated Tiparp^fl/flCre^Alb mice (Figure 9B). Only 7 metabolites were significantly changed in vehicle-treated Tiparp^fl/flCre^Alb compared with Tiparp^fl/fl mice, showing that the loss of TIPARP had little effect on basal liver metabolism (Supplementary Table 4). Consistent with previous studies, dioxin treatment resulted in significant lipidomic changes, with accumulation of several classes of free fatty acids and metabolites linked to complex lipid homeostasis (Nault et al., 2016b). Increased lipids were observed in both the Tiparp^fl/fl and Tiparp^fl/flCre^Alb animals, with a few classes of lipids that were differentially expressed in the 2 groups (Supplementary Tables 2 and 3). These included certain long-chain acylcarnitines, fatty acid dicarboxylates, and complex lipids such as plasmalogens and sphingolipids. However, 129 metabolites were altered in dioxin-treated Tiparp^fl/flCre^Alb compared with Tiparp^fl/fl mice (Table 1; Supplementary Table 5). Altered metabolite levels in Tiparp^fl/flCre^Alb and Tiparp^fl/fl mice were analyzed for pathway overrepresentation (enrichment) and connectivity within related metabolites (impact) using MetaboAnalyst (Xia and Wishart, 2016) (Figs. 9C and 9D). Only glycerophospholipid metabolism was common among the top 5 or 6 significant pathways (Supplementary Tables 6 and 7). Dioxin-treated Tiparp^fl/flCre^Alb mice also differed with regard to increased γ-glutamyl-ε-lysine levels (12.5-fold; Figure 9E), which may reflect increased transglutaminase activity, and polyamine metabolism (N-acetylputrescine; Figure 9F, and putrescine; Figure 9G). Because dioxin toxicity has been tightly linked with NAD⁺ levels, its precursors and metabolites, and because TIPARP activity is dependent on NAD⁺, we examined NAD⁺ metabolism (Diani-Moore et al., 2010, 2017; He et al., 2013). Dioxin-dependent increases in nicotinamide ribonucleoside (Figure 9H) and nicotinamide (Figure 9I) were observed in Tiparp^fl/flCre^Alb and Tiparp^fl/fl mice, respectively. Significant decreases in intrahepatic NAD⁺ levels were only observed in dioxin-treated Tiparp^fl/flCre^Alb mice (Figure 9J).

Figure 9.

Dioxin-induced hepatic metabolomic disruption. A, PCA of hepatic metabolomic analysis after 3-day treatment of Tiparp^fl/fl (fl/fl) and Tiparp^fl/flCre^Alb with CO or 10 μg/kg dioxin. B, Venn diagram of the overlapping metabolites that were significantly altered between dioxin treated Tiparp^fl/fl and Tiparp^fl/flCre^Alb. Metabolic pathway enrichment analysis of altered hepatic metabolites (p < .05) in dioxin-treated Tiparp^fl/flCre^Alb (C) and Tiparp^fl/fl mice (D). Hepatic levels of gamma-glutamyl-epsilon lysine (E), N-acetylputrescine (F), putrescine (G), nicotinamde ribonucleoside (H), nicotinamide (I), and NAD⁺ (J). Data represent the mean ± SEM (n = 6–8).

Table 1.

A Summary of the Numbers of Biochemicals That Achieved Statistical Significance (p ≤ .05) Among the Different Comparisons

	Statistical Comparisons
ANOVA contrasts	$\frac{{\text{Tiparp}}^{\text{fl}/\text{fl}}\text{TCDD}}{{\text{Tiparp}}^{\text{fl}/\text{fl}}\text{CO}}$	$\frac{{\text{Tiparp}}^{\text{fl}/\text{fl}}{\text{Cre}}^{\text{Alb}}\text{TCDD}}{{\text{Tiparp}}^{\text{fl}/\text{fl}}{\text{Cre}}^{\text{Alb}}\text{CO}}$	$\frac{{\text{Tiparp}}^{\text{fl}/\text{fl}}{\text{Cre}}^{\text{Alb}}\text{CO}}{{\text{Tiparp}}^{\text{fl}/\text{fl}}\text{CO}}$	$\frac{{\text{Tiparp}}^{\text{fl}/\text{fl}}{\text{Cre}}^{\text{Alb}}\text{TCDD}}{{\text{Tiparp}}^{\text{fl}/\text{fl}}\text{TCDD}}$
Total biochemicals (p< .05)	124	213	7	129
Biochemicals (up | down )	75 | 49	163 | 50	4 | 3	111 | 18

DISCUSSION

We previously reported that TIPARP acts as part of negative feedback loop to regulate AHR activity, and that global loss of TIPARP expression increases sensitivity to dioxin-induced toxicities such as steatohepatitis and wasting syndrome (Ahmed et al., 2015; MacPherson et al., 2013). Since hepatocyte-specific deletion of AHR prevents dioxin-induced hepatotoxicity, we reasoned that hepatocyte-specific deletion of TIPARP would result in increased dioxin-induced hepatotoxicity. We therefore generated a hepatocyte-specific TIPARP deletion (Tiparp^fl/flCre^Alb) mouse strain. We also generated a whole-body knockout TIPARP (Tiparp^Ex3−/−) strain in which Tiparp is deleted by the removal of exon 3, making it distinct from other TIPARP null lines (Ahmed et al., 2015; Kozaki et al., 2017; Schmahl et al., 2007). Here we show that Tiparp^Ex3−/− and Tiparp^fl/flCre^Alb mice are both more sensitive than WT mice to dioxin-induced hepatotoxicity and lethality. These findings provide further support for the importance of TIPARP in AHR signaling and for its role in protecting against dioxin-induced toxicity (Ahmed et al., 2015; Matthews, 2017), as well as demonstrating that the expression of TIPARP in hepatocytes plays a key role in the manifestations of this toxicity.

Tiparp^fl/flCre^Alb mice treated with dioxin lost significant body weight without any reduction in food intake. Hepatic glycogen and epididymal WAT levels were also reduced, pointing to a possible deficiency in the efficiency of intestinal nutrient absorption resulting in altered metabolism. Dioxin-induced hypophagia contributes to body weight and adipose tissue loss in many species, but numerous studies using pair-feeding or total parenteral nutrition have failed to identify a single explanation for the weight loss (Linden et al., 2010; Seefeld et al., 1984). The severe hepatotoxicity and extensive hepatosteatosis in dioxin-treated Tiparp^fl/flCre^Alb and Tiparp^Ex3−/− mice, would cause impaired liver function that could result in reduced intestinal nutrient absorption and impaired liver homeostasis (Kalaitzakis, 2014).

Dioxin-treated Tiparp^fl/flCre^Alb and Tiparp^Ex3−/− mice exhibit many of the alterations in lipid homeostasis, increased hepatic inflammation and other toxic endpoints that have been reported in other studies (Boverhof et al., 2006; Duval et al., 2017). Tiparp^fl/flCre^Alb and Tiparp^Ex3−/− mice exhibited increased hepatosteatosis due to increased expression of genes regulating lipid uptake (Cd36), but decreased expression of those involved in fatty acid β-oxidation and de novo lipogenesis (Scd1, Srebp1, Ppara) (Ahmed et al., 2015; Duval et al., 2017; Lee et al., 2010). Scd1 expression was increased in Tiparp^fl/fl mice, but decreased in Tiparp^fl/flCre^Alb mice. One possible explanation is that the increased hepatosteatosis in Tiparp^fl/flCre^Alb mice results in negative regulation of Scd1 by other factors or hormones (Mauvoisin and Mounier, 2011). Srebp1 expression levels were decreased in Tiparp^fl/flCre^Alb and Tiparp^Ex3−/− mice, supporting findings from a recent high-dose dioxin exposure study (Duval et al., 2017). Srebp1 expression is positively regulated by the liver X receptor (LXR) and PPARα. TIPARP is an LXR coactivator, so the loss of Tiparp expression combined with the reduced PPARα expression levels could also contribute to reduced SREBP1 levels (Bindesboll et al., 2016).

CYP7A1 plays a critical role in the control of bile acid and cholesterol homeostasis as the rate-limiting enzyme in the classic bile acid synthesis pathway (Gupta et al., 2001). Overexpression of mouse CYP7A1 protects against high-fat diet induced obesity, fatty liver and insulin resistance (Li et al., 2010), whereas in humans, genetic deficiency of CYP7A1 leads to hyperlipidemia (Pullinger et al., 2002). The decrease in CYP7A1 expression levels in treated Tiparp^fl/flCre^Alb and Tiparp^Ex3−/− mice is in agreement with studies of male C57BL/6 mice treated with 0.01 to 30 μg/kg dioxin every 4 for 28 days (Fader et al., 2017), and could be a contributing factor to the increased hepatosteatosis and reduced WAT levels observed, through reduced lipid absorption resulting from altered bile acid homeostasis (Fader et al., 2017). In support of this, we observed increased hepatic levels of taurochenodeoxycholic acid and taurocholic acid in dioxin-treated Tiparp^fl/flCre^Alb mice but not in Tiparp^fl/fl mice.

Metabolomic profiling studies identified significant changes in metabolite levels in dioxin-treated Tiparp^fl/flCre^Alb mice compared with Tiparp^fl/fl mice. Many of the major treatment-based changes were conserved in the 2 cohorts (ie, accumulated fatty acids, changes related to nucleotides and polyamines). However, the extent of change of the affected metabolites differed between the Tiparp^fl/fl and Tiparp^fl/flCre^Alb groups. Dioxin-treated Tiparp^fl/flCre^Alb mice, however, exhibited significant increases in gamma-glutamyl-epsilon lysine levels compared with their untreated counterparts or dioxin-treated Tiparp^fl/fl mice. This metabolite is formed by tissue transglutaminase (TG2), which catalyzes crosslinks between glutamine and lysine residues of proteins (Iismaa et al., 2009). TG2 activity increases following acute and chronic liver injury, and aberrant TG2 activation has been implicated in the development of fibrosis and cancer (Iismaa et al., 2009). In contrast, retinoid-induced TG2 mRNA up-regulation is reduced by dioxin treatment in a human squamous cell carcinoma cell line (Krig and Rice, 2000). The increased γ-glutamyl-ε-lysine observed in Tiparp^fl/flCre^Alb mice suggests that TIPARP might influence TG2 activity following dioxin-induced liver damage.

We observed that the expression of most of the AHR target genes examined was increased in response to dioxin in Tiparp^fl/flCre^Alb and Tiparp^fl/fl compared with WT mice, including Cxcl2 (macrophage inflammation protein-2) and Serpine1 genes (Son and Rozman, 2002). CXCL2 is a member of the CXC subfamily of chemokines that are crucial for neutrophil recruitment to sites of inflammation following hepatic injury (Marra and Tacke, 2014). Levels of PAI-1, the product of Serpine1 gene, were higher in Tiparp^fl/flCre^Alb mice than in Tiparp^fl/fl mice. PAI-1 is a physiologic inhibitor of plasminogen activators that regulate fibrosis via regulation of the extracellular matrix. PAI-1 expression is increased in dioxin-induced fibrosis (Nault et al., 2016a).

The mRNA levels of AHR repressor (AHRR), a negative regulator of AHR (Mimura et al., 1999), were increased in Tiparp^fl/flCre^Alb compared with Tiparp^fl/fl mice. AHRR is a potent inhibitor of AHR activity in vitro (Karchner et al., 2009) that exhibits gene- and tissue-specific inhibition of AHR signaling in mice (Hosoya et al., 2008). Although the effect of Ahrr loss on dioxin-induced wasting syndrome has not been reported, AHRR transgenic mice are protected from dioxin-induced lethality and hepatotoxicity (Vogel et al., 2016).

Increased TIPARP and PARP1 activity have been proposed to be important in augmenting dioxin toxicity through the depletion of NAD⁺. Indeed, NAD⁺ repletion or treatment with the pan-PARP inhibitor PJ34 can prevent dioxin-induced thymic atrophy and hepatosteatosis in a chicken embryo model (Diani-Moore et al., 2017). However, we observed decreased hepatic NAD⁺ levels in dioxin-treated Tiparp^fl/flCre^Alb mice, suggesting that PARP1 or an NAD⁺-consuming enzyme other than TIPARP is responsible for the reduced NAD⁺ levels after dioxin treatment in mice. Another possible explanation is that there are species differences in the AHR-TIPARP signaling axis, such that TIPARP protects against dioxin toxicity in mice but enhances it in avian species. Further studies using gene targeting methods to delete TIPARP in nonmurine models are needed to explain these discrepancies.

AHR is also a key regulator of gut homeostasis, inflammation, immunity and T-cell differentiation (Stockinger et al., 2014). AHR is required for the maintenance of intraepithelial lymphocytes, which are the first line of immune defense in the intestine. Loss of AHR or reduced exposure to dietary AHR ligands compromises these cells, leading to increased microbial load, immune activation and epithelial damage (Li et al., 2011). Moreover, AHR activation by dietary indoles (indole-3-carbinol) improves colitis and protects against experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis (Lamas et al., 2016; Li et al., 2011; Monteleone et al., 2011; Rouse et al., 2013). An unanswered question is whether TIPARP also regulates endogenous AHR signaling and if so, how would its loss affect the protective role of the AHR signaling pathway in models of inflammatory disease? Kynurenine, an endogenous AHR ligand, was reported to repress type-I interferon responses during viral infection in an AHR- and TIPARP-dependent manner, supporting the notion that TIPARP has a broad role in AHR biology and regulates endogenous ligand-induced AHR activation (Yamada et al., 2016).

In summary, we provide evidence from 2 additional mouse models that the loss of TIPARP expression increases sensitivity to dioxin toxicity and lethality. Hepatocyte-specific loss of TIPARP is sufficient to increase sensitivity to dioxin hepatotoxicity, steatosis and lethality, highlighting the importance of liver damage in the dioxin-induced wasting syndrome. Our results provide further support for the importance of the AHR-TIPARP axis in regulating dioxin toxicity, and potentially in regulating the biological actions of AHR following its activation by endogenous or dietary ligands.

SUPPLEMENTARY DATA

Supplementary data are available at Toxicological Sciences online.

FUNDING

This work was supported by Canadian Institutes of Health Research (CIHR) operating grants (MOP-494265 and MOP-125919), CIHR New Investigator Award, an Early Researcher Award from the Ontario Ministry of Innovation (ER10-07-028), an unrestricted research grant from the DOW Chemical Company, the Johan Throne Holst Foundation, Novo Nordic Foundation and the Norwegian Cancer Society to J.M.