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

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.


Introduction
The liver is a highly sexually dimorphic organ.There is increasing evidence for sexually-dimorphic regulation of xenobiotic clearance, responses to drugs and drug-induced liver injury.We postulated that the mechanisms underlying such dimorphism may involve the constitutive androstane receptor (CAR, NR1I3).CAR is a liver-enriched member of the nuclear receptor superfamily that controls ligand-dependent regulation of gene expression.Upon ligand-binding, CAR translocates to the nucleus, heterodimerises with retinoid X receptor a, and binds the xenobiotic response element located on DNA, upstream of the promoter sequences of its target genes.CAR was first described as a xenobiotic receptor that recognises a wide variety of drugs, foods, and environmental pollutants. 1,2Later studies then unveiled that CAR can also be activated by endobiotics such as bilirubin, bile acids, and steroid hormones. 3,4Upon activation, CAR regulates the expression of critical enzymes involved in phase I, II, and III xenobiotic metabolism pathways, 5,6 thereby playing a central role in xenobiotic detoxification and clearance.Moreover, CAR is involved in glucose and lipid homeostasis, although its exact role in hepatic metabolism remains controversial. 7epatic expression of Car and its main target genes Cyp2b9 and Cyp2b10 is higher in female mice than in males. 8,9Moreover, treatment with nonylphenol, a moderate CAR activator, induced expression of cytochromes P450 (CYPs) more strongly in the female mouse liver than in males. 10Similarly, treatment with 1,4-bis [2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), the prototypical pharmacological agonist of murine CAR (mCar), 11 increased liver proliferation in female more than in male mice. 12Our previous study showed that deletion of CAR expression had a stronger impact on female hepatic gene expression than on males; however, CAR-deleted males developed spontaneous steatosis during ageing while females did not. 13Therefore, CAR is thought to impact rodent liver gene expression in a sex-dependent manner, with higher CAR activity and higher sensitivity to CAR activation in females.Interestingly, in humans, the expression and activity of CYP2B6, the prototypical target gene for human CAR (hCAR), were higher in the liver of women compared with men, indicating that sexual dimorphism of CAR activity was also transposable to humans. 8espite the recognition of its sexually dimorphic activity, in vivo studies conducted so far on both male and female mice have focused on the impact of CAR activation on the regulation of CYPs, 14 on genes involved in cell cycle and hepatocarcinogenesis, 15,16 or on long non-coding RNA. 17A genome-wide comparison of the effects of CAR activation in male and female mice is still lacking.
In this study, we used hepatic microarray and metabolomics analysis of wild-type (Car +/+ ) and whole-body knockout littermate (Car -/-) male and female mice treated with either Corn oil (CO, vehicle) or TCPOBOP to elucidate the potential sexdependent impact of CAR activation.

Animal models
In vivo studies were performed in a conventional laboratory animal room following the European Union Guidelines for Use and Care of Laboratory Animals.This project was approved by an independent ethics committee (CEEA-86 Toxcométhique, authorisation number 2019123014045837).The animals were treated humanely with due consideration to the alleviation of distress and discomfort.All mice were housed at 22 C ± 2 C on a 12-h light (ZT0-ZT12) 12-h dark (ZT12-ZT24) cycle, where ZT indicates Zeitgeber time; ZT0 is defined as the time when the lights are turned on.Animals were allowed free access to food (Teklad Global 18% Protein Rodent Diet) and tap water.Car -/- mice (backcrossed on the C57BL/6J) were engineered by Wei et al. 1 and were bred for 15 years in our animal facility.Car +/- mice were co-bred and gave birth to true Car +/+ and Car -/- littermate mice, which were then separated by sex and genotype at 4 weeks of age and were randomly allocated to the different experimental groups.Nine-week-old male and female mice included in TCPOBOP groups received a daily intraperitoneal injection of 1,4-bis [2-(3,5-dichloropyridyloxy)]benzene (TCPO-BOP, Sigma Aldrich) diluted in CO used as vehicle at 3 mg/kg for 4 days, whereas CO mice received CO only (Sigma Aldrich).One cage housing n = 6 mice per group was used.At ZT16 (6 h after the last TCPOBOP injection), mice were anaesthetised with isoflurane and xylazine (2%, 2 mg/kg) then blood from the vena cava was collected into lithium heparin-coated tubes (BD Microtainer, Franklin Lake, NJ, USA).Plasma was prepared by centrifugation (1,500×g, 15 min, 4 C) and stored at −80 C. Following euthanasia by cervical dislocation, the liver and perigonadal white adipose tissue were removed, weighted and snap-frozen in liquid nitrogen, and then stored at −80 C until use.
To confirm Fmo3 regulation upon TCPOBOP treatment an independent experiment was conducted using the same experimental groups but with different timing of TCPOBOP treatment leading to the same total dose of TCPOBOP: intraperitoneal injection either with TCPOBOP diluted in CO at 3 mg/kg every 2 days for 10 days or with CO, at ZT10.Mice were euthanised by cervical dislocation at ZT8 and liver was removed, weighed and snap-frozen in liquid nitrogen and stored at −80 C.
For thrombus formation analysis, another set of 7-week-old C57BL/6J mice were purchased from Charles River laboratories, acclimatised for 2 weeks, then randomly allocated to the different experimental groups: Female Corn Oil (F CO, n = 10), female TCPOBOP (F TCPOBOP, n = 10), male Corn Oil (M CO, n = 10), male TCPOBOP (M TCPOBOP, n = 10) (two cages per group).Then, mice were fed with 1% choline-enriched diet (D13090101, Research Diets) for 10 days.Mice included in the TCPOBOP groups received an intraperitoneal injection of TCPOBOP diluted in CO at 3 mg/kg for the last 4 days of diet, whereas CO mice received CO only, at ZT0. Between ZT3 and ZT8 whole blood was drawn from the inferior vena cava of anaesthetised mice (100 mg/kg ketamine, 10 mg/kg xylazine) into heparin sodium (10 IU/ml) and mice were euthanised by cervical dislocation and liver was removed, weighed and snap-frozen in liquid nitrogen and stored at −80 C until use.

Gene expression
Gene expression profiles were performed at the GeT-TRiX facility (GénoToul, Génopole Toulouse Midi-Pyrénées, Toulouse, France) using Agilent Sureprint G3 Mouse GE v2 microarrays (8 × 60K, design 074809) following the manufacturer's instructions.Data acquisition and statistical analyses were performed as previously described. 18A correction for multiple testing was applied using the Benjamini-Hochberg procedure to control the false discovery rate (FDR).Probes with fold change (FC) > − 1.5 and FDR < − 0.05 were considered to be differentially expressed between conditions.The enrichment of Gene Ontology (GO) biological processes was evaluated using Metascape. 19Data are available in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE228554.
For real-time quantitative polymerase chain reaction (RT-qPCR), 2 lg RNA samples were reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA).Table S1 presents the SYBR Green assay primers.Amplifications were performed using an ABI Prism 7300 Real-Time PCR System (Applied Biosystems).RT-qPCR data were normalised to TATA-box-binding protein (Tbp) mRNA levels.
Proton nuclear magnetic resonance ( 1 H-NMR)-based metabolomics Plasma samples and liver polar extracts were prepared and analysed using 1 H-NMR-based metabolomics.All spectra were obtained on a Bruker DRX-600-Avance NMR spectrometer (Bruker) on the AXIOM metabolomics platform (MetaToul).Details on experimental procedures, data pre-treatment and statistical analysis were described previously. 18Parameters of the final discriminating orthogonal projection on latent structurediscriminant analysis (O-PLS-DA) are indicated in the figure legends.To identify metabolites responsible for discrimination between the groups, the O-PLS-DA correlation coefficients (r 2 ) were calculated for each variable.Correlation coefficients above the threshold defined by Pearson's critical correlation coefficient (p <0.05; |r| >0.7; for n = 6 per group) were considered significant.For illustration purposes, the area under the curve of several signals of interest was integrated and significance tested with two-way ANOVA as described below.For metabolite identification 1 H- 13 C heteronuclear single quantum coherence (HSQC) spectra were obtained on one representative sample for each biological matrix.Lists of metabolites measured are presented in Table S2 and S3.

Multi-omics analyses
Bidirectional correlations between plasma metabolites and hepatic transcripts were investigated using N-integration discriminant analysis with DIABLO, an algorithm that aims to identify a highly correlated multi-omics signature discriminating several experimental groups using the R package Mixomics v6.10.9. 20e used two components in the models, and for the estimation of model parameters, the cross-validation procedure method was used.For the correlation networks, only correlations with a Spearman's rank correlation coefficient >0.96 were plotted.

Analyses of plasma markers
Alanine aminotransferase (ALT), phosphatase alkaline (ALP), total cholesterol, high-density lipoprotein (HDL-cholesterol), triglycerides, and free fatty acids were determined using an ABX Pentra 400 biochemical analyser (Anexplo facility, Toulouse, France).Blood glucose levels were measured from the vena cava with an AccuCheck Performa glucometer (Roche Diagnostics).Values for Fmo3 gene expression were calculated using the GEO2R tool for microarray data and using GREIN 21 for RNA sequencing data.
Thrombi formation under flow Biochips microcapillaries (Vena8Fluoro+, Cellix) were coated with a collagen fibril suspension (50 lg/ml) and saturated with a solution of 0.5% bovine serum albumin in phosphate-buffered saline without Ca 2+ /Mg 2+ .Mouse blood was transferred into heparin (10 IU/ml), and DIOC6 (2 lM) was used to label platelets in whole blood.Using a syringe pump (PHD-2000; Harvard Apparatus) to apply a negative pressure, labelled blood was then perfused through a microcapillary for indicated time at a wall shear rate of 1,500 s -1 (67.5 dynes/cm 2 ).Thrombus formation was visualised with an ×40 oil immersion objective for both fluorescent and transmitted light microscopy; the light source was provided by Colibri (Zeiss) and was recorded in real-time (one frame every 20 s).Thrombi volumes were calculated by thresholding of the surface covered by thrombi on a slice of Zstack images using IMARIS software. 22her statistical analyses All univariate statistical analyses were performed using Graph-Pad Prism v.9 (GraphPad Software, San Diego, CA, USA).Outliers were identified using the ROUT method.The Kolmogorov-Smirnov test of normality was applied to all data.Two-way ANOVA was performed within each genotype using sex (male or female) and treatment (CO or TCPOBOP) as fitting factors for the models and a p value representing interactions were reported.If p sex*treatment was significant, Sidak's multiple comparisons test was used as a post-hoc test to determine which group differed from its appropriate control, otherwise p values representing the main effects from the ANOVA model (namely sex or treatment) were reported.For platelet aggregation measures, a mixed-effect model was fitted using time and treatment as fixed effects.A p <0.05 was considered significant.Results are given as the mean ± SEM.

Results
Analysis of hepatic transcripts revealed a majority of sexindependent CAR-target genes upon TCPOBOP treatment To investigate the potential sex-dependent consequences of CAR activation, we treated Car +/+ and Car -/-male and female mice with TCPOBOP (Fig. 1A).TCPOBOP did not affect the total body, decreased perigonadal white adipose tissue and increased liver weights in Car +/+ male and female mice (Fig. 1B).We confirmed Car deletion and observed increased expression of Cyp2b10 and Cyp2c55, two prototypical CAR target genes, in TCPOBOP-treated Car +/+ males and females.Cyp2c55 induction by TCPOBOP was significantly higher in females (Fig. 1C).We characterised the impact of CAR activation by TCPOBOP on hepatic gene expression using microarrays.TCPOBOP was a very specific CAR agonist since there was no significantly regulated gene in Car -/-male mice, and only two in Car -/-female mice (Fig. S1), we thus continued our analysis using Car +/+ mice only.Principal component analysis (PCA) of the entire expression data set from Car +/+ mice revealed that individuals clustered separately according to treatment on the first axis and to sex on the second axis (Fig. 1D), illustrating a major effect of CAR activation on the liver transcripts.Comparison of the lists of differentially expressed genes (DEGs) upon TCPOBOP treatment in males vs. females Car +/+ demonstrated that more than half of TCPOBOP-modulated genes were common to males and females (Fig. 1E and Fig. S2).Using all 4663 TCPOBOP-sensitive genes, we highlighted two gene clusters that exhibited sex-independent responses (Fig. 1F).Genes upregulated upon TCPOBOP (cluster 1, 2,366 genes) were mainly involved in the 'cell cycle' (p = 10 -46 ) and 'cellular response to xenobiotic stimulus' (p = 10 -25 , Fig. 1F and Table S4B), whereas downregulated genes (cluster 2, 2297 genes) were enriched for 'carboxylic acid catabolic process' (p = 10 -17 ) and 'regulation of lipid metabolic process' (p = 10 -14 , Fig. 1F and Table S4D).We focused on well-described CAR target genes involved in the cell cycle (Fig. 1G) and carbohydrate and lipid metabolism (Fig. 1H) and confirmed that regulation upon TCPOBOP treatment was similar in both males and females (Table S5).-Log 10 (p) 14  16   Cluster 1 enrichment analysis (2,366 genes)       Role of CAR in sexually-dimorphic regulation of hepatic gene expression in response to TCPOBOP TCPOBOP impacted a much higher number of genes in the liver of females vs. males (40% more DEGs in females vs. males, Fig. 1E).Accordingly, PCA of the microarray data projected on the second and third principal components showed a distinct clustering of TCPOBOP-and CO-treated females, whereas males from both groups were merged (Fig. 2A).To identify genes with sexdependent regulation upon TCPOBOP, we next focused on the DEGs with a significant interaction between sex and treatment (Table S6).These 486 sex-specific DEGs clustered within four distinct expression profiles (Fig. 2B and C).Female-specific upregulated genes (cluster 1, 215 genes) were involved in xenobiotic metabolism (p = 10 -12 ) and extracellular matrix remodelling (p = 10 -6 ) and contained genes encoding for collagens (Col4a1), extracellular matrix degrading metalloproteinases (Mmp12, Mmp13) and proteinases involved in the processing of procollagens (Adamst2, Adamst4, Adamst14, and Adamst15), whereas female-specific downregulated genes (cluster 3, 170 genes) were involved in phase I xenobiotic metabolism (p = 10 -8 ) and flavin monooxygenase (FMO)-dependent oxidations (p = 10 - 6 ) (Fig. 2D-F).As indicated above, male-specific CAR target genes were fewer.The male-specific upregulated genes (cluster 4, 37 genes) were involved in 'steroid metabolism' (p = 10 -5.5 ), whereas no significantly enriched metabolic pathway was found using the male-specific downregulated genes (cluster 2, 64 genes) (Fig. 2D and E and Table S6).Overall, this analysis provided evidence that 10% of the TCPOBOP-sensitive genes were regulated in a sexdependent manner (Fig. 2G).

CAR regulated plasma lipoprotein metabolism in a sexindependent manner
We next explored the systemic consequences of CAR activation using plasma metabolomics.PCA analysis of the whole plasma metabolic profiles showed a separation of male vs. female mice on the first principal component, illustrating a constitutive sexual dimorphism in plasma metabolite levels, while TCPOBOPtreated Car +/+ were discriminated from Car -/-mice and from Car +/+ CO-treated mice on the second principal component, illustrating a significant effect of TCPOBOP on plasma metabolites (Fig. 3A).As seen for hepatic transcripts, there was no significant differences between metabolic profiles from TCPOBOP vs. CO-treated Car -/-mice (Fig. S3).In Car +/+ males, TCPOBOP treatment decreased glucose, increased lactate levels, and had a major impact on circulating lipoproteins: cholesterol and several broad lipid peaks were strongly decreased upon TCPOBOP treatment, while other lipid peaks were increased (Fig. 3B).In Car +/+ females, cholesterol and lipid signals were changed in a similar manner than in males (Fig. 3C).Putative assignment of these differential peaks revealed that the decreased cholesterol peak reflected HDL-cholesterol, decreased lipid peaks belonged to LDLs and increased lipids belonged to VLDLs.The area under the curve for selected HDL-cholesterol, lipid-LDL and lipid-VLDL signals further illustrated this CAR-dependent impact of TCPO-BOP treatment on circulating lipoproteins in both sexes (Fig. 3D).Total plasma-, HDL-cholesterol, plasma triglycerides, free fatty acids, and glucose were quantified through additional classical biochemical assays and confirmed the strong CAR-dependent impact of TCPOBOP on plasma metabolites observed by metabolomics (Fig. S4).
Plasma metabolomics and hepatic transcriptomic data statistical integration revealed a strong correlation between plasma metabolites and liver transcripts (R 2 = 0.94) regardless of sex (Fig. 3E).The correlation network highlighted a strong positive correlation between Apoc1 mRNA and plasma LDL-cholesterol and HDL-cholesterol (Fig. 3F).This led us to investigate more closely the hepatic expression of genes involved in hepatic cholesterol metabolism (Fig. 3G).CAR activation significantly decreased the expression of most apolipoproteins.Expression of the Ldl receptor (Ldlr), which is responsible for LDL clearance was unchanged; however, the expression of Vld receptor (Vldlr) was increased by a factor of four upon TCPOBOP treatment.Mttp, the protein that transports triglycerides and cholesterol esters in the endoplasmic reticulum for VLDL synthesis was also significantly increased.Moreover, TCPOBOP treatment impacted cholesterol and bile acid metabolism with decreased expression of genes involved in cholesterol transport, decreased expression of genes involved in bile acid synthesis and increased expression of genes involved in bile acid detoxification and transport.All significant changes in hepatic mRNA and plasma metabolites related to cholesterol metabolism were CAR-dependent and were similar in both sexes (Fig. S5 and S6).Altogether, these results illustrate that CAR activation deeply modulates hepatic and systemic cholesterol metabolism in a sex-independent way.

TCPOBOP induced liver oxidative stress and toxicity in a sexbiased way
We next performed metabolic profiling of hydrophilic metabolites in liver tissue.PCA of the entire metabolic profile revealed a distinct clustering of Car +/+ mice treated with TCPOBOP vs. all other mouse groups on the first principal component, whereas male and female mice were separated on the second component, revealing once again a major impact of CAR activation on liver metabolites (Fig. 4A).TCPOBOP-treated males displayed significant changes in hepatic levels of many amino-acids (increased glutamine and glutamate and decreased valine, leucine, and isoleucine), energy-related metabolites (increased lactate and 3hydroxybutyrate, and decreased succinate), cell membrane constituents (decreased choline and glycerophosphocholine and increased phosphocholine) and metabolites involved in oxidative stress (decreased hypotaurine) (Fig. 4B).In females, most of these metabolites followed the same pattern, with perturbations of metabolites involved in oxidative stress being more pronounced than in males (significant increased levels of reduced [GSH], oxidised [GSSG], and total [Gsx] glutathione) (Fig. 4C).AUC for glutathione signals illustrated that TCPOBOP induced a more pronounced hepatic oxidative stress in females than in males (Fig. 4D).All significant changes in hepatic metabolites related to oxidative stress upon TCPOBOP were CAR-dependent (Fig. S7).Finally, sex-dependent TCPOBOP hepatic toxicity was confirmed through biochemical quantification of plasmatic markers (Fig. 4E-F).Circulating levels of ALT were significantly higher in TCPOBOP-treated Car +/+ females vs. males, whereas plasma ALP was significantly increased in both sexes upon = 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.
TCPOBOP with a tendency to higher levels in males (P sex*treatment = 0.12).Overall, our results demonstrate a sex-dependent impact of CAR activation on liver metabolism and on the toxicity profile.

CAR modulated liver trimethylamine metabolism through regulation of Fmo3 gene expression mostly in females
Another intriguing sex-dependent hepatic impact of TCPOBOP was the female-specific, CAR-dependent increased level of trimethylamine (TMA) (Fig. 4C and Fig. 5A).TMA is a gut microbiota-dependent metabolite that is metabolised to TMAO by the liver-specific flavin monooxygenase 3 (FMO3). 23This result was in accordance with the female-specific decrease of Fmo3 mRNA expression observed previously in the microarray data (Fig. 2E) and was confirmed here with RT-qPCR (Fig. 5B).Finally, we quantified hepatic TMAO and confirmed a twofold decrease of this metabolite in TCPOBOP-treated Car +/+ females compared with vehicle-treated females (Fig. 5C).Female-biased inhibition of Fmo3 mRNA by CAR activation was reproducible in an independent study in which mice were treated with TCPOBOP or CO every 2 days for 10 days (Fig. 5D).Next, we analysed Fmo3 hepatic expression in several publicly available gene expression datasets.In the first experiment, Car -/-mice were knocked-in with human CAR coding sequence 24 and were fed diets containing 0 (CTRL) or 1,000 ppm PB, an indirect activator of both mCAR and hCAR. 25We found that PB-fed hCAR mice had significantly lower expression of hepatic Fmo3 mRNA compared with control mice (Fig. 5E).The second study compared wild-type (WT) mice treated with TCPOBOP vs. COtreated mice and hCAR mice treated with CITCO (a specific agonist of hCAR) vs. CO-treated hCAR mice. 26Both TCPOBOP and CITCO-treated mice had significantly lower Fmo3 hepatic mRNA compared with their relative controls (Fig. 5F).Finally, the last two studies were conducted in chimeric mice with human hepatocytes treated with PB. 25,27 Again, we found a significant decrease in Fmo3 hepatic gene expression in response to PB in both datasets (Fig. 5G and 5H).It is worth noting that all publicly available studies were conducted in male mice only, which could explain why the decrease in Fmo3 expression seen upon CAR activation by PB or CITCO was of lower magnitude than that seen in our own in vivo experiments in females.Finally, we took advantage of the only available ChIP-seq analysis of hCAR binding in vivo to date 28 and observed that, among the 6,364 unique genes associated with high-confidence hCAR-binding genes, Fmo3 was found as a hCAR-direct binding gene with a p value = 1.26 × 10 -29 (Fig. 5I).Thus, in females, CAR activation by TCPOBOP perturbated the metabolism of TMAO from TMA by inhibiting the expression of Fmo3 (Fig. 5J) and this result might be relevant to humans.
CAR activation decreased platelet hyperactivity induced by dietary choline supplementation FMO3 activity and TMAO levels have been shown to modulate platelet hyper-responsiveness and thrombosis potential. 29,30We thus wondered whether CAR activation could also influence platelet function.To enhance platelet responsiveness, female and male mice were fed a choline-enriched diet before treatment with TCPOBOP (Fig. 5K).We confirmed significant hepatic CAR activation in both sexes (Fig. 5L) and examined thrombi formation.Whole blood from female mice treated with TCPOBOP formed smaller thrombi over time compared with blood from control mice.In males, TCPOBOP treatment did not affect thrombi formation.Thus, in vivo TCPOBOP treatment modulates platelet activation and reduces the thrombotic risk of females specifically (Fig. 5M and N).

Discussion
The liver appears to be one of the most sexually dimorphic organs and expression of genes involved in drug metabolism is sexually dimorphic in rodents and humans. 8,31CAR is the target of many drugs and is widely involved in the control of expression of xenobiotic metabolising genes.However, a genome-wide description of sex-specific CAR-dependent sensitive genes was lacking.Here, we provide an exhaustive study of the transcriptomic impact of acute pharmacological CAR activation in male vs. female mice and novel insights into the metabolic impact of this activation.
First, most TCPOBOP-modulated genes were regulated in a similar manner in male and female livers, especially those involved in the cell cycle.3][34] The underlying mechanisms depend on direct CAR-dependent induction of Mdm2, a primary inhibitor of P53 and induction of the transcription factor FoxM1, 35 which is essential for the initiation of carcinogen-induced liver tumours, thus resulting in modulation of many genes implicated in cell proliferation, cellular growth, apoptosis, and cell differentiation, such as Gadd45b, Ccnd1, Ccnb1, C-myc, and Yap. 35,36Few studies have highlighted that sex could influence TCPOBOP-induced liver proliferation but showed inconsistent results.Some studies described that female mice were more sensitive to TCPOBOPinduced liver proliferation, 12,37 whereas others showed no tumour development in female mice treated with the genotoxin diethylnitrosamine followed by TCPOBOP, compared with males. 38Similarly, after a single injection of TCPOBOP, male mice displayed a deeper disturbance of key cell cycle genes. 16Unlike these studies, we did not reveal any sexual dimorphism on hepatomegaly and cell cycle gene modulation upon CAR activation.However, a long-term analysis of TCPOBOP-induced liver tumours conducted in male and female mice in parallel would be required to further investigate this discrepancy.
We next observed a strong impact of TCPOBOP treatment on plasma lipoproteins, with decreased total-, HDL-and LDLcholesterol measured in both sexes.This result is consistent with previous findings whereby TCPOBOP decreased circulating levels of plasma HDL in both WT and transgenic mice expressing human apolipoprotein A-1, at least in part through downregulation of ApoA-1 hepatic gene expression. 39Similarly, in Ldlr -/-mice fed a Western-diet, TCPOBOP decreased circulating levels of ApoB-containing lipoproteins (mainly VLDL and LDL) and reduced the development of atherosclerotic lesions, 40 presumably through CAR-mediated induction of the VLDL receptor, a receptor involved in the clearance of VLDL and LDL as a backup for the LDL receptor. 41Here, we confirm that CAR activation results in decreased ApoA-1 and increased Vldlr hepatic mRNA levels, which could both participate to the observed decrease of plasmatic HDL and LDL levels.Moreover, we also observed a strong decrease in hepatic expression of other major lipoproteincoding genes (namely ApoB, Apoc1, and ApoE), and of Lecithin cholesterol acyl transferase (Lcat, another constitutive component of HDL) which could also play a role.
Another well-known function of CAR is its ability to promote bile acid detoxification during cholestasis. 42,43As previously described, we found that the expression of genes involved in hydroxylation, sulfation, and excretion of bile acids was significantly enhanced upon CAR activation, whereas expression of genes involved in bile acid synthesis and cholesterol transport was decreased in both sexes.The emerging role of CAR in cholesterol homeostasis represents new perspectives in the treatment of hypercholesterolaemia and atherosclerosis. 39,44The current study confirms and extends these previous studies reporting the effects of TCPOBOP on hepatic expression of genes involved in bile acid, cholesterol and lipoprotein metabolism, as well as those on lipoprotein concentrations, are sexindependent, at least in mice.Our findings may have clinical relevance.Indeed, a recent study combining genome-wide analysis of cholestatic mice genetic models and data-mining of human patient cohorts with various liver diseases unravelled a significant enrichment of CAR-sensitive genes in cholestatic livers specifically. 45Moreover, CAR activation in cholestasis leads to alterations of drug metabolism with significant effects on drug-induced hepatotoxicity.Drug-induced liver injury (DILI) is still a serious clinical concern and one of the most common drug adverse reactions.DILI clinical phenotype is influenced by age and sex. 46,47Here, we found that, upon TCPOBOP, 385 genes displayed a female-specific vs. 101 genes with a male-specific response.Many female-specific genes were involved in extracellular matrix organisation.We also observed stronger perturbations of hepatic metabolites involved in glutathione metabolism in livers of females compared with males, reflecting higher hepatic oxidative stress.Finally, we highlighted a sexually dimorphic impact of CAR activation on clinical markers of liver toxicity, namely significantly higher levels of ALT in females compared with males, and a trend toward higher ALP levels in males.This result is consistent with the sex-influence on DILI clinical phenotype with cytolytic damage being more frequently observed in women, whereas cholestatic damage presented a male predominance. 46,47It is well known that women experience higher rates, 48 and more severe 49 adverse drug reactions than men.However, mechanistic explanations for these observations are often lacking.Our present results suggest that drugs interacting with CAR may be considered with particular attention before their use in women.
Limitations of our study include the use of only one drug (TCPOBOP), whereas DILI has been shown to depend both on patient characteristics and on drug properties. 50Our results therefore need to be confirmed with other drugs that act as CAR agonists.
Another novel finding from our study was the strong increase in hepatic TMA upon TCPOBOP administration observed in female mice specifically.TMA is a product of the gut microbial metabolism of phosphatidylcholine, choline, and L-carnitine.It is transported from the gut to the liver via the portal vein and Noxidised into TMAO by host FMO3. 51Analysis of natural genetic variation in inbred strains of mice indicate that FMO3 and TMAO are significantly correlated and explain more than 10% of the variation in atherosclerosis. 51Since then, it has been confirmed that high circulating levels of TMAO are linked to increased thrombotic and cardiovascular risks in animal and human studies, even after adjustment for known cardiovascular risk factors. 52,53Consistent with increased TMA, hepatic Fmo3 mRNA expression and TMAO concentration were both strongly decreased in Car +/+ females treated with TCPOBOP.We confirmed the CAR-dependent regulation of Fmo3 mRNA in publicly available cohorts that used different hCAR models and different mCAR and hCAR agonists, therefore suggesting that the regulation of Fmo3 expression is not dependent on the CAR agonist used and might be relevant in humans.In rodents, hepatic Fmo3 knockdown was sufficient to decrease dietdependent platelet responsiveness and thrombotic potential. 29,30Here, we observed that, in conditions of diet-induced platelet hyper-responsiveness, CAR activation was indeed sufficient to significantly modulate thrombus growth in female mice specifically.We postulate that this effect is, at least in part, attributable to the CAR-mediated downregulation of Fmo3 expression and activity in females.Nowadays, drugs represent the main cause of platelet dysfunction. 54,55Our results suggest that drugs or other xenobiotics (such as pollutants, foods) that interact with CAR could decrease thrombus formation in a prothrombotic context.These compounds may provide a beneficial effect by modulating platelet activation and thrombosis.We, therefore, highlight a new axis between hepatic xenobiotic metabolism and blood haemostasis.We suggest that this axis may be especially relevant in women.However, there are important species-specific sex-based differences in FMO3 expression: its expression is female-specific in mice as a result of modulation by sex steroids, 56 whereas its abundance was significantly associated with females in humans but to a much lower extent than in rodents. 57Thus, the gender-specificity and clinical relevance of this CAR-FMO3-TMAO-platelet axis deserves further investigation.In addition to platelet function and thrombotic risk, an increase in the TMAO plasma concentration has also been shown to increase the risk of impaired glucose tolerance, 58 colorectal cancer, 59 chronic kidney disease, 60 and overall mortality. 61Whether drugs interacting with CAR could also influence these TMAO-dependent endpoints deserves further investigations.
In summary, the present study provides an exhaustive description of the sex-independent and sex-dependent CARsensitive genes and demonstrates a stronger impact of CAR pharmacological activation on hepatic transcriptome and metabolism of the female.Additionally, CAR activation impacted the TMA-FMO3-TMAO pathway in females, which might link drugs and environmental xenobiotic exposure with platelet aggregation and other TMAO-sensitive physiological responses.
:Cellular response to xenobiotic stimulus GO:0009725:Response to hormone GO:0032787: Monocarboxylic acid metabolic process GO:0046395:Carboxylic acid catabolic process GO:0044283:Small molecule biosynthetic process GO:0010564:Regulation of cell cycle GO:0006259:DNA metabolic process GO:0006259:Positive regulation of cell cycle GO:0019216:Regulation of lipid metabolic process