Pharmacological activation of constitutive androstane receptor induces female-specific modulation of hepatic metabolism

Abstract

Background & aims: The constitutive androstane receptor (CAR) is a nuclear receptor that binds diverse xenobiotics and whose activation leads to the modulation of the expression of target genes involved in xenobiotic detoxification and energy metabolism. Although CAR hepatic activity is considered to be higher in women than in men, its sex-dependent response to an acute pharmacological activation has seldom been investigated.

Methods: The hepatic transcriptome, plasma markers, and hepatic metabolome, were analysed in Car^+/+ and Car^-/- male and female mice treated either with the CAR-specific agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or with vehicle.

Results: Although 90% of TCPOBOP-sensitive genes were modulated in a sex-independent manner, the remaining 10% showed almost exclusive female liver specificity. These female-specific CAR-sensitive genes were mainly involved in xenobiotic metabolism, inflammation, and extracellular matrix organisation. CAR activation also induced higher hepatic oxidative stress and hepatocyte cytolysis in females than in males. Hepatic expression of flavin monooxygenase 3 (Fmo3) was almost abolished and was associated with a decrease in hepatic trimethylamine-N-oxide (TMAO) concentration in TCPOBOP-treated females. In line with a potential role in the control of TMAO homeostasis, CAR activation decreased platelet hyper-responsiveness in female mice supplemented with dietary choline.

Conclusions: More than 10% of CAR-sensitive genes are sex-specific and influence hepatic and systemic responses such as platelet aggregation. CAR activation may be an important mechanism of sexually-dimorphic drug-induced liver injury.

Impact and implications: CAR is activated by many drugs and pollutants. Its pharmacological activation had a stronger impact on hepatic gene expression and metabolism in females than in males, and had a specific impact on liver toxicity and trimethylamine metabolism. Sexual dimorphism should be considered when testing and/or prescribing xenobiotics known to activate CAR.

Keywords: Hepatic xenobiotic metabolism; Lipoprotein metabolism; Platelet aggregation; Sexual dimorphism; Trimethylamine-N-oxide.

Graphical abstract

Fig. 1

Modulation of classical CAR-controlled pathways is sex-independent. (A) Experimental design. (B) Body weight, perigonadal WAT, and liver weights. (C) Hepatic gene expression. Results are given as the mean ± SEM. ∗Treatment effect, ^#sex effect. ∗ or ^#p <0.05, ∗∗ or ^##p <0.01, ∗∗∗ or ^###p <0.001 (two-way ANOVA). (D) PCA of the whole liver transcriptomic dataset in Car^+/+ mice. (E) Venn diagram representing the number of genes significantly modulated by TCPOBOP in the liver of Car^+/+ male and female mice (p_adj<0.05 and fold-change >1.5). (F) Hierarchical clustering and pathway enrichment analysis using the 4,663 genes significantly regulated upon TCPOBOP in males or females (p_adj <0.05 and fold-change >1.5). (G) Heatmaps representing the log(fold-change) of gene expression between TCPOBOP- and CO-treated Car^+/+ males and females and (H) for genes involved in cell cycle. and carbohydrate and lipid metabolism. CAR, constitutive androstane receptor; CO, corn oil; PCA, principal component analysis; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene; WAT, white adipose tissue.

Fig. 2

Identification of sex-dependent CAR-sensitive genes. (A) PCA of the liver transcriptomic dataset in Car^+/+mice. (B) Hierarchical clustering using the 486 genes with significant p_{sex∗treatment}(p_adj <0.05 and fold-change>1.5). (C) Gene expression profiles in each cluster. (D) Pathway enrichment analysis. (E) Heatmaps representing the log(fold-change) of gene expression between TCPOBOP- and CO-treated Car^+/+ males and females for the top 10 genes in each cluster. (F) Hepatic gene expression of genes involved in inflammation and fibrosis derived from microarray data. ∗Treatment effect, ^#sex effect. ∗ or^#p_adj <0.05, ∗∗ or^##p_adj <0.01, ∗∗∗ or^###p_adj <0.001 (linear models). (G) Genes modulated by TCPOBOP treatment in a sex-dependent and -independent way. CAR, constitutive androstane receptor; CO, corn oil; PCA, principal component analysis; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene.

Fig. 3

Sex-independent impact of CAR activation on lipoprotein metabolism. (A) PCA of plasma metabolomic dataset. (B) Coefficient plots related to the PLS-DA models discriminating between plasma spectra from TCPOBOP vs. CO Car^+/+ males. Parameters of the PLS-DA model: Q²Y = 0.84, p = 0.001. (C) Coefficient plots related to the PLS-DA models discriminating between plasma spectra from TCPOBOP vs. CO Car^+/+ females. Parameters of the PLS-DA model: Q²Y = 0.89, p = 0.001. (D) AUC of the ¹H-NMR spectra was integrated for glucose, lactate, cholesterol, lipid CH₃(CH₂)_n (mainly in Ldl), lipid CH₃CH₂CH₂C = (mainly in Vldl) signals. Results are given as the mean ± SEM. ∗Treatment effect, ^#sex effect. ∗ or ^#p <0.05, ∗∗ or ^##p <0.01, ∗∗∗ or ^###p <0.001 (two-way ANOVA). (E) Multi-omic integrative analysis performed on plasma metabolomic and hepatic transcriptomic datasets (DIABLO model). (F) Correlation network between hepatic transcripts and plasma metabolites (R² > 0.97, DIABLO model). (G) Fold-change (TCPOBOP- vs. CO-treated Car^+/+ mice) of hepatic expression for genes involved in lipoprotein metabolism. ∗Treatment effect, ^#sex effect. ∗p_adj <0.05, ∗∗p_adj <0.01, ∗∗∗p_adj <0.001 (linear model). CAR, constitutive androstane receptor; CO, corn oil; PB, phenobarbital; PCA, principal component analysis; PLS-DA, orthogonal projection on latent structure-discriminant analysis; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene; Vldr, very low-density protein receptor.

Fig. 4

TCPOBOP treatment increases liver oxidative stress and toxicity in a sex-biased manner. (A) PCA of the liver metabolomic dataset. (B) Coefficient plots related to the PLS-DA models discriminating liver extract spectra from TCPOBOP- and CO-treated Car^+/+ males. Parameters of the PLS-DA model: Q²Y = 0.89, p = 0.001. (C) Coefficient plots related to the O-PLS-DA models discriminating between liver extract spectra from TCPOBOP- and CO-treated Car^+/+ females. Parameters of the PLS-DA model: Q²Y = 0.91, p = 0.001. (D) AUC of the ¹H-NMR spectra was integrated for the glutathione signals (GSH, reduced form; GSSG, oxidised form; GSx, total glutathione) and for hypotaurine. (E) Plasma alanine aminotransferase. (F) Plasma alkaline phosphatase. Results are given as the mean ± SEM. ∗Treatment effect, ^#sex effect. ∗ or^#p <0.05, ∗∗ or ^##p <0.01, ∗∗∗ or^###p <0.001 (two-way ANOVA). CAR, constitutive androstane receptor; CO, corn oil; GSH, reduced glutathione; GSSG, oxidized glutathione; GSx; PLS-DA, projection on latent structure-discriminant analysis; PCA, principal component analysis; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene.

Fig. 5

CAR modulates liver TMA metabolism by regulating Fmo3 gene expression, mostly in females. (A) AUC of the ¹H-NMR spectra for TMA. (B) Hepatic gene expression. (C) Hepatic content of TMAO. (D) Hepatic gene expression in independent confirmatory experiment. Results are mean ± SEM. ∗Treatment effect, ^#sex effect. ∗ or ^#p <0.05, ∗∗ or ^##p <0.01, ∗∗∗ or ^###p <0.001 (two-way ANOVA). (E–H) Hepatic mRNA expression derived from publicly available datasets. ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 (one-way ANOVA or Student t test). (I) Proposed model for direct binding of hCAR to Fmo3 regulatory DNA sequences based on Niu et al. (created with BioRender.com). (J) Proposed impact of CAR activation on TMA metabolism (created with BioRender.com). (K) Experimental design. (L) Hepatic gene expression. Results are mean ± SEM. ∗Treatment effect, ^#sex effect. ∗ or ^#p <0.05, ∗∗ or ^##p <0.01, ∗∗∗ or ^###p <0.001 (two-way ANOVA). (M) Representative images of platelet adhesion. (N) Quantification of platelet adhesion to a microfluidic chip surface ∗p <0.05, ∗∗p <0.01 (one-way ANOVA). CAR, constitutive androstane receptor; GSH, reduced glutathione; GSSG, oxidised glutathione; GSx, total glutathione; PCA, principal component analysis; PLS-DA, orthogonal projection on latent structure-discriminant analysis; TMA, trimethylamine; TMAO, trimethylamine N-oxide.