Constitutive Androstane Receptor Agonist Initiates Metabolic Activity Required for Hepatocyte Proliferation.

Activation of the constitutive androstane receptor (CAR, NR1I3) by chemical compounds induces liver hyperplasia in rodents. 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a mouse CAR agonist, is most often used to study chemically induced liver hyperplasia and hepatocyte proliferation in vivo. TCPOBOP is a potent murine liver chemical mitogen, which induces rapid liver hyperplasia in mice independently of liver injury. In recent years, great amount of data has been accumulated on the transcription program that characterizes the TCPOBOP-induced hepatocyte proliferation. However, there are only few data about the metabolic requirements of hepatocytes that divide upon exposure to xenobiotics. In the present study, we have employed liquid chromatography - mass spectrometry technology combined with statistical analysis to investigate metabolite profile of small biomolecules, in order to identify key metabolic changes in the male mouse liver tissue after TCPOBOP administration. Analysis of biochemical pathways of the differentially affected metabolites in the mouse liver demonstrated significant TCPOBOP-mediated enrichment of several processes including those associated with nucleotide metabolism, amino acid metabolism, and energy substrate metabolism. Our findings provide evidence to support the conclusion that the CAR agonist, TCPOBOP, initiates an intracellular program that promotes global coordinated metabolic activities required for hepatocyte proliferation. Our metabolic data might provide novel insight into the biological mechanisms that occur during the TCPOBOP-induced hepatocyte proliferation in mice.

Discovery of Novel Human Constitutive Androstane Receptor Agonists with the Imidazo[1,2-a]pyridine Structure.

The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.

The role of mouse and human peroxisome proliferator-activated receptor-α in modulating the hepatic effects of perfluorooctane sulfonate in mice.

Perfluorooctane sulfonate (PFOS) is a stable environmental contaminant that can activate peroxisome proliferator-activated receptor alpha (PPARα). In the present work, the specific role of mouse and human PPARα in mediating the hepatic effects of PFOS was examined in short-term studies using wild type, Ppara-null and PPARA-humanized mice. Mice fed 0.006 % PFOS for seven days (∼10 mg/kg/day), or 0.003 % PFOS for twenty-eight days (∼5 mg/kg/day), exhibited higher liver and serum PFOS concentrations compared to controls. Relative liver weights were also higher following exposure to dietary PFOS in all three genotypes as compared vehicle fed control groups. Histopathological examination of liver sections from mice treated for twenty-eight days with 0.003 % PFOS revealed a phenotype consistent with peroxisome proliferation, in wild-type and PPARA-humanized mice that was not observed in Ppara-null mice. With both exposures, expression of the PPARα target genes, Acox1, Cyp4a10, was significantly increased in wild type mice but not in Ppara-null or PPARA-humanized mice. By contrast, expression of the constitutive androstane receptor (CAR) target gene, Cyp2b10, and the pregnane X receptor (PXR) target gene, Cyp3a11, were higher in response to PFOS administration in all three genotypes compared to controls for both exposure periods. These results indicate that mouse PPARα can be activated in the liver by PFOS causing increased expression of Acox1, Cyp4a10 and histopathological changes in the liver. While histopathological analyses indicated the presence of mouse PPARα-dependent hepatic peroxisome proliferation in wild-type (a response associated with activation of PPARα) and a similar phenotype in PPARA-humanized mice, the lack of increased Acox1 and Cyp4a10 mRNA by PFOS in PPARA-humanized mice indicates that the human PPARα was not as responsive to PFOS as mouse PPARα with this dose regimen. Moreover, results indicate that hepatomegaly caused by PFOS does not require mouse or human PPARα and could be due to effects induced by activation of CAR and/or PXR.

Genomic comparisons between hepatocarcinogenic and non-hepatocarcinogenic organophosphate insecticides in the mouse liver.

Short-term biomarkers of toxicity have an increasingly important role in the screening and prioritization of new chemicals. In this study, we examined early indicators of liver toxicity for three reference organophosphate (OP) chemicals, which are among the most widely used insecticides in the world. The OP methidathion was previously shown to increase the incidence of liver toxicity, including hepatocellular tumors, in male mice. To provide insights into the adverse outcome pathway (AOP) that underlies these tumors, effects of methidathion in the male mouse liver were examined after 7 and 28 day exposures and compared to those of two other OPs that either do not increase (fenthion) or possibly suppress liver cancer (parathion) in mice. None of the chemicals caused increases in liver weight/body weight or histopathological changes in the liver. Parathion decreased liver cell proliferation after 7 and 28 days while the other chemicals had no effects. There was no evidence for hepatotoxicity in any of the treatment groups. Full-genome microarray analysis of the livers from the 7 and 28 day treatments demonstrated that methidathion and fenthion regulated a large number of overlapping genes, while parathion regulated a unique set of genes. Examination of cytochrome P450 enzyme activities and use of predictive gene expression biomarkers found no consistent evidence for activation of AhR, CAR, PXR, or PPARα. Parathion suppressed the male-specific gene expression pattern through STAT5b, similar to genetic and dietary conditions that decrease liver tumor incidence in mice. Overall, these findings indicate that methidathion causes liver cancer by a mechanism that does not involve common mechanisms of liver cancer induction.

Off-target lipid metabolism disruption by the mouse constitutive androstane receptor ligand TCPOBOP in humanized mice.

The constitutive androstane receptor (CAR) controls xenobiotic clearance, regulates liver glucose, lipid metabolism, and energy homeostasis. These functions have been mainly discovered using the prototypical mouse-specific CAR ligand TCPOBOP in wild-type or CAR null mice. However, TCPOBOP is reported to result in some off-target metabolic effects in CAR null mice. In this study, we compared the metabolic effects of TCPOBOP using lipidomic, transcriptomic, and proteomic analyses in wild-type and humanized CAR-PXR-CYP3A4/3A7 mice. In the model, human CAR retains its constitutive activity in metabolism regulation; however, it is not activated by TCPOBOB. Notably, we observed that TCPOBOP affected lipid homeostasis by elevating serum and liver triglyceride levels and promoted hepatocyte hypertrophy in humanized CAR mice. Hepatic lipidomic analysis revealed a significant accumulation of triglycerides and decrease of its metabolites in humanized CAR mice. RNA-seq analysis has shown divergent gene expression levels in wild-type and humanized CAR mice. Gene expression regulation in humanized mice is mainly involved in lipid metabolic processes and in the PPAR, leptin, thyroid, and circadian clock pathways. In contrast, CAR activation by TCPOBOP in wild-type mice reduced liver and plasma triglyceride levels and induced a typical transcriptomic proliferative response in the liver. In summary, we identified TCPOBOP as a disruptor of lipid metabolism in humanized CAR mice. The divergent effects of TCPOBOP in humanized mice in comparison with the prototypical CAR-mediated response in WT mice warrant the use of appropriate model ligands and humanized animal models during the testing of endocrine disruption and the characterization of adverse outcome pathways.