In 2024, the FDA granted approval for resmetirom, a selective but moderately potent agonist of the thyroid hormone receptor (THR) β, marking the first drug for the treatment of metabolic dysfunction-associated steatohepatitis (MASH) in the US. Given the absence of selectivity data, we conducted a comprehensive screening against other nuclear receptors implicated in metabolic regulation, in addition to THRβ. Reporter gene assays revealed that resmetirom also binds several off-target nuclear receptors, including constitutive androstane receptor (CAR), retinoic acid receptor-related orphan receptors (RORs), and hepatocyte nuclear factor 4α (HNF4α). However, subsequent in vitro experiments, designed to better recapitulate physiological conditions, showed that THRβ modulation is functionally dominant. These preliminary findings suggest that pharmacotherapeutic efficacy of resmetirom remains intact despite the identified off-target effects, supporting its clinical application in the treatment of MASH.

Endocrine-disrupting chemicals (EDCs) are increasingly implicated in the development of metabolic disorders such as type 2 diabetes mellitus (T2DM). As hepatic dysfunction is a hallmark of early metabolic disease, we investigated how EDCs may contribute to glucose dysregulation using human HepaRG cells. Ten EDCs-bisphenol A (BPA), bisphenol F (BPF), bisphenol S (BPS), cadmium chloride (CdCl2, 1µM), butylparaben (BP), 1,1-dichloro-2,2-bis(4-chlorophenyl)ethene (p,p'-DDE), dibutyl phthalate (DBP), di(2-ethylhexyl) phthalate (DEHP), perfluorooctanoic acid (PFOA), and perfluorooctanesulfonic acid (PFOS)-were tested at 25µM for 5days. We assessed multiple endpoints related to carbohydrate metabolism, including gene expression, mitochondrial function, glycogen content, glucose export, glycolytic capacity, and lactate release. Among the tested compounds, p,p'-DDE induced the most pronounced metabolic disruption, significantly reducing glycogen storage, glycolytic capacity, lactate export, and the expression of key genes involved in glucose metabolism. Using luciferase-based reporter cell lines, p,p'-DDE was found to activate primarily the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR). However, siRNA-mediated knockdown of these receptors did not reverse the changes induced by p,p'-DDE in gene expression, suggesting a more complex or alternative mechanism of action. These findings demonstrate that p,p'-DDE perturbs hepatic carbohydrate metabolism and may contribute to the pathogenesis of T2DM, highlighting the need for further mechanistic investigation.

Letter to the editor regarding the review article: Yamada T, Cohen SM, Lake BG. The modes of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR) and the peroxisome proliferator-activated receptor alpha (PPARα) are not relevant to human cancer risk: an updated critical evaluation. Crit Rev Toxicol. 2025;55(5):549–586. doi: 10.1080/10408444.2025.2513332

Response to the Letter from Professor C. R. Wolf and colleagues concerning CRT, 55: 549–586, 2025: The modes of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR) and the peroxisome-proliferator-activated receptor alpha (PPARα) are not relevant to human cancer risk: an updated critical evaluation

Exposure to a persistent organic pollutant mixture resulted in sex-specific steatotic liver disease: Role of the liver-endocrine axis.

Binding modes and interaction mechanism of bisphenol A and its analogs in constitutive androstane receptor

Mode of action analysis for mouse liver tumor formation by MGK-264 and human relevance.

Multiple prenatal adverse environmental factors alter hepatic cytochrome P450 (CYP) enzymes expression in offspring, with these changes persisting after birth. These factors induce fetal exposure to excessive maternal glucocorticoids (GCs), however, the mechanisms by which intrauterine GCs exposure programs offspring CYP expression remain unclear. Given that GCs are high‐affinity ligands for the glucocorticoid receptor (GR), this work employs dexamethasone (DEX), a GR agonist, to establish a prenatal dexamethasone exposure (PDE) model for investigating the role of GR activation in CYPs programming. The model is implemented to pregnant Wistar rats and heterozygous liver‐specific GR knockout mice. Results show that PDE consistently increases the expression of hepatic CYP3A1 and CYP2B1, thereby enhancing the metabolic enzyme efficiency in adult male offspring. In vitro experiment further validates that GC‐induced activation of GR increases the binding of P300/cAMP response element‐binding protein (CBP) to the promoter region of constitutive androstane receptor (CAR), which leads to sustained H3K9 and H3K27 acetylation at the CAR locus, indirectly promoting CYP expression in adult offspring. Conclusively, the GR‐CAR pathway may play a pivotal role in programming CYP3A alteration in offspring exposed to elevated intrauterine GCs. This study provides novel insights into individual variations of CYPs expression and metabolic patterns.

Synthetic pyrethroids, such as permethrin, are commonly used insecticides in crop agriculture, clothing items such as camping and military uniforms, and for treating insect infestations in people and pets. Growing epidemiological evidence supports an association between pyrethroid insecticide exposures and cardiovascular diseases risk, including coronary heart disease. Studies in rats show that pyrethroid insecticides are pro-atherogenic, in part, by increasing circulating total LDL-C and oxidized LDL-C and cholesterol accumulation in the aorta. Here, we used the ApoE Null mouse model to test the hypothesis that chronic exposure to permethrin promotes atherosclerosis. Male and female mice were fed a custom diet based on the "What we eat in America” analysis (NHANES). Cholesterol was included at 296 mg per 1000 kcal (i.e., the 90 th percentile of cholesterol intake in the typical American diet). The mice were exposed to vehicle or 10, 50 and 500 µg/kg/day doses of permethrin delivered in food for 11-12 weeks. By comparison, the Environmental Protection Agency identifies a maximum daily exposure of 50 µg permethrin/kg/day as protective of adverse effects. Using allometric scaling, the approximate human-equivalent doses were 1, 4 and 41 µg/kg/day. For comparison, US military personnel wearing permethrin-treated uniforms have an estimated average daily dose of permethrin of 3-9 µg/kg/day. We observed that at a daily dose as low as 50 μg permethrin/kg for 11-12 weeks, atherosclerotic plaque area was larger in permethrin-exposed compared to vehicle-exposed mice. While the effect of permethrin on plaque area was not different between sexes, we observed a greater reduction in weight gain in female mice and a greater increase in liver lipids in male mice at 500 μg/kg/day. No differences in serum liver enzymes (i.e., AST and ALT) were observed. Given that pyrethroid insecticides have been shown to activate several nuclear receptors in vitro , we analyzed total liver RNA for the expression of target genes of peroxisome proliferator activated receptor α, constitutive androstane receptor and pregnane X receptor, however no significant differences across groups were observed. Our results demonstrate that chronic exposure to pyrethroid insecticides accelerates atherosclerosis in murine models that may represent a mechanism for the clinical epidemiological observations linking pyrethroids to increased cardiovascular disease risk.

Constitutive androstane receptor activation modulates YAP expression and activity through ubiquitination and sumoylation.

Distinct responses of the constitutive androstane receptor NR1I3 to indole-containing metabolites of bacterial origin.

Comparative study of CYP450 gene regulation in human pluripotent stem cell-derived liver 2D cell and 3D organoid models.

Cytochrome P450 (P450) enzymes in the small intestine play a critical role in determining the systemic availability of orally administered drugs. In dogs, the major intestinal drug-metabolizing P450 enzymes are CYP3A98, an intestine-specific isoform, and CYP2B11, which are expressed in both the liver and intestines. This study aimed to establish differentiated canine duodenal organoids and evaluate the expression, inducibility, and enzymatic activity of these key intestinal P450 enzymes. Duodenal organoids were generated from healthy canine intestinal biopsies and cultured under expansion and differentiation conditions. CYP3A98 and CYP2B11 gene expression was assessed by quantitative reverse transcription polymerase chain reaction, while enzyme function was evaluated using midazolam (CYP3A98) and bupropion (CYP2B11) hydroxylation assays. To assess P450 induction, organoids were treated with rifampicin (a pregnane X receptor [PXR] selective inducer) and phenobarbital (a constitutive androstane receptor [CAR] inducer). Organoid differentiation significantly upregulated CYP3A98 and CYP2B11 mRNA expression and enzyme activity. Rifampicin (50 μM) strongly induced CYP3A98 gene expression (7.1-fold) and enzyme activity (2.5-fold) without affecting CYP2B11 expression. CYP3A98 and CYP2B11 expression were unaffected by phenobarbital treatment at a CAR-selective concentration (250 μM). However, treatment with phenobarbital at a high concentration (2 mM), known to directly bind and activate PXR, resulted in a significant increase in CYP3A98 expression (3.6-fold) and activity (1.4-fold) without substantially affecting CYP2B11 expression. Differentiated canine duodenal organoids expressed functional CYP3A98 and CYP2B11. CYP3A98 was inducible through PXR, while CYP2B11 was not regulated by CAR or PXR. This platform may provide a valuable tool for evaluating drug absorption, metabolism, and drug–drug interactions in veterinary drug development.Significance StatementA physiologic canine intestinal in vitro model for drug development is lacking in veterinary medicine. The canine differentiated duodenal organoids used in this study expressed CYP3A98 and CYP2B11 enzymes and may provide a physiological platform for studying drug metabolism and drug–drug interactions during the development of veterinary pharmaceuticals.

Glutathione (GSH) is a potent antioxidant regulating oxidative stress, but whether exogenous GSH supplementation mitigates stress injury through host-microbiome and liver interactions remains unclear. This study aimed to determine the regulatory mechanism of GSH using in vivo (28-day-old weaned piglets) and in vitro (alpha mouse liver 12 (AML12) cells) stress injury models. Thirty-five healthy weaned piglets (mean body weight (9.52±0.20) kg) were fed diets supplemented with 0.01%, 0.03%, or 0.06% GSH for 4 weeks, followed by being injected intraperitoneally with paraquat (PQ) on days 28, 30, and 32. AML12 cells exposed to tert-butyl hydroperoxide (tBHP) were used to evaluate related mechanisms, and CINPA1was used to inhibit constitutive androstane receptor (CAR) activity. Our results showed that PQ challenge induced hepatic morphological and functional impairments, accompanied by suppression of antioxidant capacity and immune function. Notably, exogenous GSH treatment significantly increased CD4+/CD8+ T lymphocyte ratio, GSH, immunoglobulin A and interleukin-10 levels in serum, reduced the secretion and gene expression of pro-inflammatory factors to alleviate liver injury. Moreover, GSH treatment significantly promoted CAR nuclear translocation and regulated the expression of detoxification genes in response to liver inflammation. Additionally, GSH treatment improved the diversity and relative abundance of colonic probiotic bacteria such as UCG_002, Christensenellaceae_R_7_group, Prevotellaceae_NK3B31_group, Prevotella, Oscillospira, and unclassified_UCG_010. Furthermore, in vitro results showed that 3 mmol L-1 GSH administration could increase the expression of CAR pathway and antioxidant related genes, and inhibit cellular ROS production in tBHP-induced AML12 cells. However, CAR inhibition by CINPA1 prevented GSH from alleviating tBHP-induced oxidative stress injury in AML12 cells. Our results indicate that exogenous GSH treatment alleviated liver injury in piglets through host-microbiome interaction and a CAR-dependent signaling pathway.

Abstract The pregnane X receptor (PXR), constitutive androstane receptor (CAR), and farnesoid X receptor (FXR) are ligand-activated nuclear receptors that modulate the hepatic metabolism of endogenous compounds, including androstenone, a testicular steroid produced by intact male pigs. Inefficient metabolism of androstenone contributes to its accumulation in adipose tissue, which promotes the development of boar taint, a meat quality issue characterized by off-odours and off-flavours in some heated pork products. Natural products (NPs) found in plants and herbal medicines act as ligands for PXR, CAR, and FXR in humans and may increase the hepatic metabolism of androstenone in boars. Therefore, this study examined the effects of several NPs, including hyperforin (HYP; PXR agonist), diallyl sulfide (DAS; CAR agonist), oleanolic acid (OA; FXR modulator), ginkgolide A (GINK; PXR and CAR agonist), and (Z)-guggulsterone (GUG; PXR agonist, CAR inverse agonist, FXR antagonist) on androstenone metabolism in porcine hepatocytes. Hepatocytes were isolated from the livers of 5-month-old crossbred [(Yorkshire x Landrace) x Duroc] boars (n=8) and incubated for 24 hours with NP treatments or dimethyl sulfoxide (DMSO) as a control. Cells were then treated with androstenone for 3 hours, after which culture media was collected for high-performance liquid chromatography analysis of androstenone metabolism and metabolite production. Hepatocytes were also harvested to assess gene expression via RT-qPCR. Statistical analysis was performed using a one-way ANOVA in SAS, with a significance threshold of p ≤ 0.05, and the Benjamini-Hochberg correction was applied to control for multiple comparisons. Relative to the DMSO control, NP treatments significantly altered the expression of key genes involved in hepatic androstenone metabolism. UGT1A6 was upregulated by GINK (p=0.05) and GUG (p=0.04) and downregulated by OA (p=0.004), while FXR expression increased with OA (p=0.02). NR2F1 was downregulated by DAS (p=0.03), GINK (p=0.03), and GUG (p=0.02), HNF4A by OA (p=0.008), and PGC1α by DAS (p=0.03) and GUG (p=0.05). NP treatments had minimal effects on overall androstenone metabolism relative to DMSO; however, treatment responses varied among individual boars. Relative to DMSO, DAS increased overall androstenone metabolism in 75% of boars, GINK, GUG, and HYP in 63%, and OA in 50%. Among positive responders, overall androstenone metabolism was significantly increased by DAS (p=0.04) and GUG (p=0.05). Additionally, DAS increased the production of Phase I androstenol metabolites (p=0.0008). These results suggest that DAS and GUG may be promising dietary treatments for boar taint but highlight the need to assess treatment responses on an individual basis. Future research should include in vivo feeding trials to evaluate the efficacy of these NPs and identify biomarkers associated with positive treatment outcomes to allow for their targeted and effective application to reduce boar taint.

Role of metabolic nuclear receptors in acute liver injury and drug-induced hepatotoxicity.

Nuclear receptors CAR and PXR as cardiometabolic regulators.

Xenobiotic-induced liver injury: Molecular mechanisms and disease progression.

P25-09 The role of Constitutive Androstane Receptor (CAR) in PFOA-induced hepatic steatosis

The constitutive androstane receptor (CAR), encoded by the Nr1i3 gene, is a nuclear receptor mainly expressed in the liver, where it regulates (xenobiotic) drug and bile acid metabolism, bilirubin clearance and energy homeostasis. CAR has emerged as a promising therapeutic target for diabetes, fatty liver disease and alcoholic liver disease, but it has barely been investigated in the context of sepsis. Since alterations in drug metabolism have been observed in sepsis patients, who may also exhibit increased serum bilirubin and bile acid levels, we hypothesize that CAR function may be impaired during sepsis. Here, we demonstrate that CAR loses its function in the liver during sepsis, as evidenced by a diminished response to its agonist TCPOBOP. We show that Nr1i3 mRNA transcription is reduced, mediated by decreased HNF4α binding to the Nr1i3 promoter and by downregulation of Ppara expression. Additionally, we show that CAR DNA binding is impaired, and we propose that HNF4α may regulate chromatin accessibility of CAR binding sites in sepsis. CAR loss-of-function further causes the downregulation of genes involved in monocarboxylic acid, fatty acid, and xenobiotic metabolism, but induces a hepatic acute phase response, which is beneficial for liver regeneration. However, CAR inhibition with CINPA1 increases sepsis lethality, associated with the further downregulation of these metabolic genes, increased upregulation of the acute phase response, but persistent downregulation of proliferation markers in the liver. Altogether, our study highlights the importance of CAR in sepsis with respect to hepatic metabolism, liver regeneration and survival. Nevertheless, CAR is unlikely to serve as a viable therapeutic target in sepsis, given its rapid downregulation and the lack of a survival benefit from TCPOBOP treatment. Instead, targeting upstream regulators such as HNF4α may represent a more effective approach.