Urinary metabolomics in Fxr-null mice reveals activated adaptive metabolic pathways upon bile acid challenge.

Farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in synthesis, metabolism, and transport of bile acids and thus plays a major role in maintaining bile acid homeostasis. In this study, metabolomic responses were investigated in urine of wild-type and Fxr-null mice fed cholic acid, an FXR ligand, using ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS). Multivariate data analysis between wild-type and Fxr-null mice on a cholic acid diet revealed that the most increased ions were metabolites of p-cresol (4-methylphenol), corticosterone, and cholic acid in Fxr-null mice. The structural identities of the above metabolites were confirmed by chemical synthesis and by comparing retention time (RT) and/or tandem mass fragmentation patterns of the urinary metabolites with the authentic standards. Tauro-3α,6,7α,12α-tetrol (3α,6,7α,12α-tetrahydroxy-5β-cholestan-26-oyltaurine), one of the most increased metabolites in Fxr-null mice on a CA diet, is a marker for efficient hydroxylation of toxic bile acids possibly through induction of Cyp3a11. A cholestatic model induced by lithocholic acid revealed that enhanced expression of Cyp3a11 is the major defense mechanism to detoxify cholestatic bile acids in Fxr-null mice. These results will be useful for identification of biomarkers for cholestasis and for determination of adaptive molecular mechanisms in cholestasis.


Animals, diets, and sample collection
Fxr -null mice and the background matched wild-type mice generated as reported previously ( 6 ) were maintained under a standard 12-h light/12-h dark cycle with water and a normal diet (NIH-31) provided ad libitum. Groups of 8-to 12-week-old male mice were put on a synthetic purifi ed diet (AIN-93G, Bio-Serv) at day 5 before treatment and maintained with water and the same diet ad libitum. The CA diet and LCA diet consisted of the control diet (AIN-93G) supplemented with 1% (w/w) CA and 0.6% (w/w) lithocholic acid, respectively. Fxr -null and wild-type mice were fed the CA diet or control diet for 7 days and the LCA diet or control diet for 4 days. Urine samples were collected from mice placed individually in metabolic cages for 24 h, before treatment and on day 6 of CA treatment or day 3 of LCA treatment. All urine samples were stored at -80°C until analyzed. At the end of the study, animals were killed, and serum and liver tissue were collected and frozen at -80°C for further analysis. Protocols for all animal studies were approved by the National Cancer Institute Animal Care and Use Committee and were carried out in accordance with the guidelines of Institute of Laboratory Animal Resources.

Serum chemistry
Serum was prepared by centrifugation at 8,000 rpm for 10 min and the catalytic activities of alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were measured in serum. Briefl y, 2 l of serum was mixed with 200 l of ALT or ALP assay buffer (Catachem, Bridgeport, CT) in a 96-well microplate and monitored at 340 nm or 405 nm for 10 min at 37°C. Serum corticosterone levels were determined using a Corticosterone EIA Kit (Cayman Chemical Co., Ann Arbor, MI) according to the manufacturer's instruction.

UPLC-TOFMS analyses
Urine aliquots were diluted with 4 vols of 50% acetonitrile and centrifuged at 18,000 g for 20 min at 4°C to remove particles and proteins. The aliquots (5 l) were injected into a reverse-phase 50 × 2.1 mm ACQUITY ® 1.7-m BEHC18 column (Waters Corp., Milford, MA) using an ACQUITY ® UPLC system (Waters) with a gradient mobile phase comprising 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). Each sample was resolved for 10 min at a fl ow rate of 0.5 ml/min with the gradient consisted of 100% A for 0.5 min, 20% B for 3.5 min, 95% B for 4 min, 100% B for 1 min, and 100% A for 1 min. The eluent was introduced by electrospray ionization into the mass spectrometer, Q-TOF Premier ® (Waters), operating in either negative ion (ESI Ϫ ) or positive ion (ESI+) electrospray ionization modes. The capillary and sampling cone voltages were set to 3000 and 30 V, respectively. The desolvation gas fl ow was set to 650 L/h and the temperature was set to 350°C. The cone gas fl ow was 50 L/h, and the source temperature was 120°C. To maintain mass accuracy, sulfadimethoxine ([M-H] -309.0658) at a concentration of 250 pg/ l in 50% acetonitrile was used as a lock mass and injected at a rate of 30 l/min. Data were acquired in centroid mode from 50 to 800 m/z in MS scanning. Tandem MS collision energy was scanned from 5 to 35 V.

Data processing and multivariate data analysis
Centroided and integrated chromatographic mass data from 50 to 800 m/z were processed by MarkerLynx ® (Waters) to generate a multivariate data matrix. Pareto-scaled MarkerLynx matri-ing that the metabolic pathways of controlling bile acid levels might be altered in Fxr -null mice ( 6 ). Recently, an adaptive response to bile acids in Fxr -null mice was reported. Loss of FXR leads to increased susceptibility to bile acid-induced liver injury and results in adaptive changes of genes involved in regulation of basolateral bile acid uptake, bile acid metabolism/detoxifi cation, and alternative basolateral bile acid secretion in an FXR-independent manner ( 7,8 ).
Metabolomics, the global approach for identifi cation and quantifi cation of metabolites in a living system or biological sample has been mostly performed using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS) combined with various multivariate data analyses ( 9 ). Recently, robust metabolomic methods using the high resolution capability of ultra-performance liquid chromatography (UPLC) coupled with the accurate mass determination of time-of-fl ight (TOF) MS were developed for detection and characterization of small organic molecules in complex biological matrices ( 10 ). A recent metabolomics study, combined with a genetically modifi ed knockout mouse, has revealed that activation of peroxisome proliferator-activated receptor ␣ (PPAR ␣ ) affects the metabolism of tryptophan, corticosterone, and fatty acids, and that novel steroid metabolites, 11 ␤ -hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11 ␤ ,20dihydroxy-3-oxopregn-4-en-21-oic acid (DHOPA), in urine can be biomarkers for activation of PPAR ␣ ( 11 ). More recently, a metabolomics study on the activation of pregnane X receptor (PXR) was performed using urine from Pxrnull mice and their congenic wild-type counterparts treated with the PXR ligand, pregnenolone 16 ␣carbonitrile ( 12 ). A novel vitamin E metabolite, ␥ -CEHC glucoside, and ␣ -CEHC glucuronide were strongly decreased upon activation of PXR and might serve as biomarkers for PXR activation. Thus, metabolomics revealed PXR-activated metabolic pathways associated with vitamin E metabolism ( 12 ). These studies highlight that metabolomics can be useful to identify small molecule signatures for gene defi ciency and activation of nuclear receptors and/or their pathophysiological stimuli.
In the present study, altered metabolomic responses were investigated in urine of Fxr -null mice administered cholic acid compared with wild-type mice. UPLC-TOFMS coupled with multivariate data analyses were used to distinguish between the urinary metabolites in Fxr -null and wild-type mice on the CA diet. The structure of novel metabolites was identifi ed and those metabolites were quantifi ed using synthetic standards. This study further revealed adaptive molecular mechanisms for activation of alternate metabolic pathways in the cholestatic Fxr -null mouse.

Reagents
Standards for taurocholate, tauro-␤ -muricholate and reagents for synthesis were purchased from Sigma (St. Louis, MO). HDOPA and DHOPA were purchased from Anbilaunch consul-analyte, and the resultant concentrations were expressed as mol/mmol creatinine (normalized).

Statistical analysis
Each group consisted of 6-12 animals for this study. All values are expressed as the means ± SD. Statistical analysis was performed by two-way ANOVA combined with Bonferroni posttests using Prism 5 (GraphPad Software Inc., San Diego, CA). Correlations between relative Cyp3a11 levels and relative abundance of taurotetrol or ALT activity were calculated using Pearson correlation tests (Prism 5). A P value of less than 0.05 was considered statistically signifi cant.

Phenotypes of Fxr -null mice
Male wild-type and Fxr -null mice fed a control diet showed no signifi cant differences in body weight, liver-tobody weight ratio, and serum ALP activity ( Table 1 ). However, after feeding 1% CA for 7 days, Fxr -null mice exhibited more severe body weight loss, higher liver-to-body weight ratios, and higher serum ALP activity than wild-type mice ( Table 1 ). These phenotypes are similar with those previously reported for wild-type and Fxr -null mice after feeding CA ( 17 ). In contrast, the typical diagnostic hepatotoxicity marker, serum ALT activity, was two-fold higher in Fxr -null mice than in wild-type mice fed control diet but was not signifi cantly changed between Fxr -null and wild-type mice ces including information on sample identity were analyzed by principal components analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) using SIMCA-P+ 12 (Umetrics, Kinnelon, NJ). To determine which ions contribute to the difference between wild-type (y = 0) and Fxr -null (y = 1) mice of the CA-fed group, orthogonal partial least squares (OPLS) was used. The loadings scatter S-plots and the contribution lists were used to describe the candidate markers that were signifi cantly different between wild-type and Fxr -null mice of CA-fed group.

Identifi cation of metabolites
To identify the structure of high-contribution score metabolites, elemental compositions were generated with MassLynx ® (Waters) based on the exact masses of the top twelve ions. To confi rm the identities of markers, authentic standards at 5-20 M in 50% acetonitrile were compared with the urine sample on the condition of MS/MS fragmentation with collision energy ramping from 15 to 35 V. Therefore, the MS/MS fragmentation spectrum of putative urine metabolites was shown to be identical to that of the authentic standards.

Synthesis of p -cresol sulfate and glucuronide
p -Cresol sulfate was prepared by treating p -cresol with chlorosulfonic acid in methylene chloride. To synthesize p -cresol glucuronide, p -cresol was reacted with tetraacetylglucuronyl trichloroimidate in the presence of boron trifl uoride diethyl etherate in methylene chloride to afford p -cresol tetraacetyl glucuronide ( 13 ). The product was hydrolyzed with 2M potassium hydroxide in methanol to obtain p -cresol glucuronide.
plot providing a clear separation between wild-type ( +/+ ) and Fxr -null ( Ϫ / Ϫ ) mice of CA-fed groups in component 1 (Y-axis) but not in the control diet group. This result revealed that more specifi c metabolic phenotypes were changed in Fxr -null mice than in wild-type mice by CA loading. After OPLS analysis of wild-type and Fxr -null mice in the CA-fed groups, a loadings S-plot showed ions with the highest confi dence and greatest contribution to separation between wild-type and Fxr -null mice in the CA-fed groups ( Fig. 1B ). The signifi cant ions increased in Fxr -null mice after CA loading were in the upper-right quadrant and those decreased were in the lower-left quadrant. The increased negative ions were labeled with a number from 1 to 12 according to the highest confi dence and greatest contribution to separation between wild-type and Fxr -null mice in CA-fed groups in Fig. 1B . Three types of metabolites, specifi cally p -cresol, corticosterone, and cholic acid metabolites, were confi rmed from the data ( Table 2 ). To identify the structure of the above ions, accurate mass values of each ion was used for chemical formula calculations using elemental composition and mass-based searches in various database/literature were performed. fed CA, which suggested that Fxr -null mice under bile acidloading conditions exhibited FXR-dependent cholestasis, but showed similar sensitivity to bile acid-induced hepatotoxicity compared with CA fed wild-type mice.

Metabolomic analysis of mouse urine
Urine samples for 0-24 h were collected before and after six days of CA feeding and analyzed by UPLC-TOFMS operating in both positive and negative ionization modes. Bile acids showed several fragment ions derived from only one bile acid in positive ion mode and thus multivariate data analysis was performed on the data matrix from the negative ionization mode. A large data matrix containing approximately 6000 ions was produced by MarkerLynx and subjected to both PCA and PLS-DA multivariate data analyses. Unsupervised PCA yielded a good separation of the data sets from the control and CA-fed groups in both wild-type and Fxr -null mice, with fi tness (R 2 value) of 0.52 and prediction power (Q 2 value) of 0.28 (data not shown). A supervised PLS-DA model with six components successfully discriminated the differences between all four groups of mice, having cumulative fi tness (R 2 X and R 2 Y values) of 0.65 and 0.96, respectively, and cumulative prediction power (Q 2 value) of 0.80. Fig. 1A shows a PLS-DA scores  Table  1 . CA, cholic acid; FXR, farnesoid X receptor; TOFMS, time-of-fl ight mass spectrometry; UPLC, ultra-performance liquid chromatography. CA-fed Fxr -null mice compared with CA-fed wild-type mice (n = 12). Conditions for UPLC-TOFMS analysis are described in "Experimental Procedures." Ion rank from a loading S-plot of OPLS analysis showed the rank of ions with the highest confi dence and greatest contribution to separation between wild-type and Fxr -null mice. Ion rank is the same as labeling of ions in Fig. 1B  ) ions correspond to tauro-3 ␣ ,6,7 ␣ ,12 ␣ -tetrol, taurocholate, cholate glucoside, tauro-7-epicholate, and taurocholate glucoside, respectively. Because the basal levels of the metabolite ions, 2, 5, 6, 10, 11, and 12 were very low, the values of fold change in each metabolite were over 50. However, the chemical structures still required experimental verifi cation using the authentic compounds.

Identifi cation and quantifi cation of p -cresol metabolites
Authentic p -cresol sulfate and p -cresol glucuronide were required to confi rm the structural identifi cation of these metabolites by UPLC-MS/MS and to quantify the levels of these metabolites.  ( Fig. 2B ). The major daughter ions 175 and 107 in negative ion mode were interpreted in the inlaid structural diagram. Because of the insuffi cient purity of synthetic standards, proper calibration curves could not be constructed for both p -cresol sulfate and p -cresol glucuronide. Therefore, relative abundance of p -cresol sulfate and p -cresol glucuronide were measured in each urine sample and expressed as response/ mmol creatinine, a variable independent of urine volume. Although the relative abundance of p -cresol sulfate and p -cresol glucuronide were decreased signifi cantly in both wild-type and Fxr -null mice after CA feeding ( P < 0.001), the wild-type mice on the CA diet exhibited a signifi cant and dramatic depletion of urinary p -cresol sulfate (8.3% of control, P < 0.001, Fig. 2C ) and p -cresol glucuronide (4.5% of control, P < 0.001, Fig. 2D ). Therefore, the Fxr -null mice on the CA diet exhibited signifi cantly greater than wildtype mice on the CA diet by 4-fold ( P < 0.01) or 10-fold ( P < 0.001) in either p -cresol sulfate or p -cresol glucuronide urine levels, respectively ( Fig. 2C, D ).

Identifi cation and quantifi cation of glucocorticoid metabolites
To identify the structure of hydroxylated HDOPA and DHOPA, authentic compounds of 19-hydroxylated HDOPA and 19-hydroxylated DHOPA were synthesized and tandem MS fragmentation was performed. The patterns of MS/MS spectra between the synthetic compounds and the hydroxylated metabolites in mouse urine were different (data not shown), suggesting that the hydroxylated HDOPA and DHOPA is not 19-hydroxyl HDOPA and 19-hydroxyl DHOPA. The identities of HDOPA and DHOPA were established by comparison of MS/MS frag-els were determined in serum ( Fig. 3E ). Indeed, Fxr -null mice showed a robust increase in corticosterone level after treatment with the CA diet (changed 24.0 ± 8.3 to 149 ± 76 ng/ml, P < 0.01 ). On the other hand, wild-type mice did not exhibit a signifi cant change in corticosterone levels (changed 13.0 ± 7.0 to 29.0 ± 17.0 ng/ml).

Identifi cation of cholic acid metabolites
Authentic cholic acid metabolites were required for elucidating structure. Tauro-3 ␣ ,7 ␤ ,12 ␣ -trihydroxycholate (tauro-7-epicholate) and 4 epimers of tauro-3 ␣ ,6( ␣ / ␤ ),7( ␣ / ␤ ),12 ␣ -tetrahydroxycholate were synthesized as described in "Experimental Procedures." However, two novel metabolites, cholate glucoside (C 30 H 50 O 10 , mass error = 3.5 ppm) and taurocholate glucoside (C 32 H 55 NO 12 S, mass mentation of authentic standards and the urinary compounds as demonstrated in the previous report ( 11 ). In addition, the concentrations of HDOPA and DHOPA in urine were determined using calibration curves. Whereas both wild-type and Fxr -null mice fed control diet had undetectable HDOPA and DHOPA in their urine, Fxr -null mice fed CA diet only had 229 ± 92 mol/mmol creatinine in HDOPA and 110 ± 25 mol/mmol creatinine in DHOPA ( Fig. 3A , B ). Moreover, the relative abundance of hydroxylated HDOPA and DHOPA was signifi cantly increased in Fxr -null mice fed CA diet as a similar pattern to the concentration of HDOPA and DHOPA ( Fig. 3C, D ). Since corticosterone metabolites are elevated in urine, serum corticosterone levels would be expected to increase in the Fxr -null mice fed CA diet, and thus corticosterone lev- fragmentation was conducted with same condition as p -cresol sulfate. C: Relative abundance of p -cresol sulfate in the 24-h urine of wild-type and Fxr -null mice treated with 1% CA or control for 7 days. Relative abundance of p -cresol sulfate (mean ± SD) was determined by the peak area responses of p -cresol sulfate normalized by the creatinine concentration in urine. D: Relative abundance of p -cresol glucuronide (mean ± SD) was determined by the peak area responses of p -cresol glucuronide normalized by the creatinine concentration in urine. Open and fi lled bars represent wild-type and Fxr -null mice groups, respectively. The P values were calculated by ANOVA with Bonferroni posttests. * P < 0.01 and ** P < 0.001 compared with wild-type mice. CA, cholic acid; FXR, farnesoid X receptor; MS/MS, tandem mass spectrometry. acid metabolites are too stable in negative ion mode to generate their tandem mass spectra ( Fig. 4 ) The identities of tauro-3 ␣ ,7 ␣ ,12 ␣ -trihydroxycholate (taurocholate), tauro-7-epicholate, and tauro-3 ␣ ,6,7 ␣ ,12 ␣tetrahydroxycholate (tauro-3 ␣ ,6,7 ␣ ,12 ␣ -tetrol) were confi rmed by comparison of retention time from authentic compounds and the urinary constituent because cholic Fig. 3. Quantifi cation of urinary corticosterone metabolites and serum corticosterone in wild-type and Fxrnull mice treated with 1% CA or control for 7 days. A: Creatinine-normalized concentration of HDOPA. B: Creatinine-normalized concentration of DHOPA. Concentrations of creatinine, HDOPA, and DHOPA were determined from calibration curve of each metabolite. All concentrations for quantifi cation were normalized to the creatinine concentration ( mol/mmol creatinine). C: Relative abundance of hydroxylated HDOPA was determined by the peak area responses of hydroxylated HDOPA normalized by the creatinine concentration in urine (response/mmol creatinine). D: Relative abundance of hydroxylated DHOPA was determined by the peak area responses of hydroxylated DHOPA normalized by the creatinine concentration in urine (response/mmol creatinine). Data were represented as mean value ± SD (n = 6-12). E: Serum corticosterone levels. The corticosterone levels were measured as described in "Experimental Procedures." Data were expressed as mean value and SD (n = 4-5). Signifi cant difference was determined by one-way ANOVA following Bonferroni's test. * P < 0.01 compared with wild-type mice. Open and fi lled bars represent wild-type and Fxr -null mice groups, respectively. CA, cholic acid; DHOPA, 11 ␤ ,20-dihydroxy-3-oxopregn-4-en-21-oic acid; FXR, farnesoid X receptor; HDOPA, 11 ␤ -hydroxy-3,20-dioxopregn-4-en-21-oic acid.

Hepatic gene expression
Metabolomic differences in urine between wild-type and Fxr -null mice after feeding the CA diet suggested that increased urinary metabolic phenotypes resulted from an adaptive response of Fxr -null mice to CA-induced toxicity through alteration of gene expression for metabolic enzymes. Specifi cally, the highly increased cholic acid metabolite in Fxr -null fed CA, tauro-3 ␣ ,6,7 ␣ ,12 ␣ -tetrol, was a 6-hydroxylated form of taurocholate, which suggested that bile acid-oxidation enzymes such as CYP3A11 and CYP2B10 were induced in Fxr -null mice ( 18 ). The hepatic expression of Cyp3a11 , a representative PXR target gene, was elevated signifi cantly by approximately 6-fold in Fxr -null mice fed both control and CA diets, compared with wild-type mice fed a control diet (data not shown). The hepatic expression of Cyp2b10 , a representative constitutive androstane receptor (CAR) target gene, was elevated by approximately 10-fold and 4-fold in Fxr -null mice fed control and CA diets, respectively, compared with wild-type mice fed a control diet (data not shown), but these differences were not statistically signifi cant. These expression levels for Cyp3a11 and Cyp2b10 were similar to a previous report ( 18 ).

Quantifi cation of cholic acid metabolites
The concentrations of taurocholate, tauro-7-epicholate, and tauro-3 ␣ ,6,7 ␣ ,12 ␣ -tetrol as well as creatinine were measured in each urine sample using dehydroxycholate as an internal standard and expressed as µmol/mmol creatinine. Urinary concentrations of taurocholate, tauro-7epicholate, and tauro-3 ␣ ,6,7 ␣ ,12 ␣ -tetrol were undetectable in both wild-type and Fxr -null mice fed control diet ( Fig. 5 ). After feeding the CA diet, taurocholate concentrations were elevated to 46.4 ± 59.5 mol/mmol creatinine in wild-type mice and to 139 ± 64.4 mol/mmol creatinine in Fxr -null mice, which were statistically significantly different ( P < 0.001). In contrast to undetectable hydroxy bile acid ([M-H] Ϫ = 530.278), which may be derived from LCA, was measured individually in addition to analysis of the relative Cyp3a11 level and serum ALT activity. A clear correlation (Pearson's correlation coeffi cient, r = 0.63, P = 0.005) was observed between individual relative abundance of the taurotetrol and Cyp3a11 expression levels ( Fig. 6A ). The relative abundance of the taurotetrol was signifi cantly 4.3-fold higher in Fxr -null than in wildtype mice ( P = 0.009). In addition, relative Cyp3a11 expression levels in liver exhibited a signifi cant increase by 3.8-fold in Fxr -null mice on the LCA diet compared with those in wild-type mice on LCA diet ( P < 0.0001). Moreover, an inverse correlation ( r = Ϫ 0.85, P < 0.0001) was observed between ALT activity and relative CYP3A11 levels in liver of both wild-type and Fxr -null mice ( Fig. 6B ). Serum ALT activity was 4860 ± 1680 IU/L in wild-type mice and 1960 ± 1090 IU/L in Fxr -null mice on LCA diet (40% lower than wild-type mice, P = 0.0003). However, serum ALP activity, a typical diagnostic cholestatic marker, was not signifi cantly different between wild-type and Fxr -null mice on LCA diet (569 ± 169 and 444 ± 195 IU/L, respectively). Taken together, these data suggested that Fxr -null mice eliminate the systemic and hepatic bile acid load more rapidly than wild-type mice by increasing the expression of CYP3A11, resulting in increased hydroxylation of bile acids and less hepatotoxicity than wild-type mice under LCA-induced cholestasis.

DISCUSSION
Global analyses of urinary metabolomes in Fxr -null mice compared with their wild-type littermates under conditions of CA loading revealed separation of three groups in a scores plot: control wild-type and Fxr -null mice; CA-fed wild-type mice; and CA-fed Fxr -null mice. These results suggest that CA feeding markedly changes urinary metabolites in Fxr -null mice and, to a lesser degree, in wild-type mice. OPLS analysis between wild-type and Fxr -null mice on the CA diet further showed that most of the increased ions were metabolites of p -cresol, corticosterone, and CA.
Urinary p -cresol comes from dietary polyphenols or tyrosine residues generated by intestinal microorganisms including Proteus vulgaris , Clostridium diffi cile , and unidentifi ed species of Lactobacillus ( 20,21 ). Urinary p -cresol was reported to be one of the dysbiosis markers, including benzoate, hippurate, phenylacetate, and hydroxybenzoate, which are associated with microbial overgrowth ( 22 ). More recently, metabolomics analysis revealed p -cresol sulfate as being unique to plasma samples of conventional mice compared with samples of germ-free mice ( 23 ). In this study, attenuated p -cresol conjugates after CA feeding suggested that oral administration of bile acids inhibits the intestinal microbial overgrowth ( 24 ). Specifi cally, CA feeding markedly attenuated urinary p -cresol metabolites in wild-type mice but to a lesser extent in Fxr -null mice, indicating that CA feeding substantially increases fecal bile acid excretion through induction of bile acid transporters in wild-type mice but to a lesser extent in Fxr -null mice, as tion reactions, some of which are catalyzed by CYP3A11, a P450 typically involved in drug metabolism ( 18 ). To characterize the effect of enhanced hydroxylation of bile acids on hepatoprotection in Fxr -null mice, LCA-induced cholestatic model was used. Because BSEP, regulated by FXR, is highly expressed in wild-type mice but not in Fxr -null mice, the CA-induced cholestasis is found only in Fxr -null mice. Therefore, administration of LCA, a hydrophobic secondary bile acid, causes intrahepatic cholestasis in wild-type mice as well as in Fxr -null mice ( 19 ). Male wild-type and Fxr -null mice were fed a diet containing 0.6% LCA for 4 days, and their urine was collected for 24 h both prior to and after 3 days on the diet. Blood and liver samples were taken for biochemical assay after 4 days on the diet. The relative abundance of a urinary taurine-conjugated tetra- Fig. 5. Quantifi cation of taurocholate, tauro-7-epicholate and tauro-3 ␣ ,6( ␣ / ␤ ),7 ␣ ,12 ␣ -tetrol in urine of wild-type and Fxr -null mice treated with 1% CA or control for 7 days. Concentrations of creatinine, taurocholate, and tauro-7-epicholate and tauro-3 ␣ ,6( ␣ / ␤ ),7 ␣ ,12 ␣ -tetrol were determined from calibration curve of each metabolite and normalized to the creatinine concentration ( mol/mmol creatinine). A: Creatinine-normalized concentration of taurocholate. B: Creatinine-normalized concentration of tauro-7-epicholate. C: Creatinine-normalized concentration of tauro-3 ␣ ,6( ␣ / ␤ ),7 ␣ ,12 ␣ -tetrol. Open and fi lled bars represent wild-type and Fxr -null mice groups, respectively. Data were represented as mean value ± SD (n = 6-12). The P values were calculated by ANOVA with Bonferroni posttests. ** P < 0.001 compared with wildtype mice. CA, cholic acid; FXR, farnesoid X receptor. activated mice, possibly mediated by induction of hydroxylation by enzymes such as CYP3A11. However, further investigation should be performed to establish the specifi c metabolic pathways of corticosterone, HDOPA, and DHOPA in cholestatic mice.
A dramatic increase of urinary corticosterone metabolites HDOPA and DHOPA, as well as serum corticosterone, was observed only in cholestatic mice (i.e., Fxr -null mice fed CA for 7 days). In cholestatic animal models and humans, the hypothalamic-pituitary-adrenal axis was activated by endotoxin and cytokines, such as tumor necrosis factor-␣ , resulting in an elevation of plasma glucocorticoid concentration ( 25 ). Most of the corticosterone present in the blood is excreted into urine and feces as more polar metabolites of corticosterone through an intensive steroid metabolism in liver and gut ( 26 ), although there has been only limited information about metabolic pathways of glucocorticoids ( 27 ). Therefore, highly increased urinary excretion of HDOPA and DHOPA in acute cholestasis may result from secretion and extensive metabolism of corticosterone. Recently, HDOPA and DHOPA were reported as highly specifi c biomarkers for activation of PPAR ␣ ( 11 ). However, high levels of HDOPA and DHOPA in CA-fed Fxr -null mice may not be associated with activation of PPAR ␣ because the expression of PPAR ␣ -target genes such as Cpt1a and Cyp4a10 were not induced by CA feeding (data not shown). Furthermore, a previous study reported that bile acids interfere with transactivation of PPAR ␣ at least in part by impairing the recruitment of transcriptional coactivators ( 28 ). Hydroxylated metabolites of HDOPA and DHOPA were also highly increased in CA-fed Fxr -null mice to an extent similar to that observed with HDOPA and DHOPA. However, an increase of these hydroxylated metabolites were not found in urine from mice treated with the PPAR ␣ agonist Wy-14,643 ( 11 ). This result indicates that CA-fed Fxr -null mice have different metabolic pathways of corticosterone from PPAR ␣ - Fig. 6. Correlation plots of hepatic Cyp3a11 expression with formation of taurotetrol or hepatotoxicity in the cholestatic mouse model induced by LCA. Fxr -null and wild-type mice were fed a 0.6% LCA diet or control diet for 4 days and urine and serum samples were collected. A: Correlation between relative CYP3A11 levels and relative abundance of taurotetrol. B: Correlation between relative CYP3A11 levels and ALT activity in serum. Relative CYP3A11 levels were determined by real-time PCR analysis using gene-specifi c primers and ␤ -actin as the internal control. Relative abundance of taurotetrol was determined by the peak area responses of taurotetrol normalized by the creatinine concentration in urine (response/mmol creatinine). ALT activities were determined as described in "Experimental Procedures." Correlation analysis was constructed in wild-type (open circle) and Fxr -null (closed circle) mice fed with LCA. Correlation coeffi cient, r values and P values were calculated by Pearson correlation test. ALT, alanine aminotransferase; FXR, farnesoid X receptor; LCA, lithocholic acid. corticosterone and CA had been highly elevated in Fxrnull mice by CA loading but not in wild-type mice. Tauro-3 ␣ ,6,7 ␣ ,12 ␣ -tetrol, one of the most increased metabolites in Fxr -null mice on a CA diet, is a marker for effi cient hydroxylation of toxic bile acids possibly by high induction of CYP3A11. Furthermore, LCA-induced toxic models proved that the enhanced expression of Cyp3a11 is the major defense mechanism to detoxify cholestatic bile acids in Fxr -null mice. These results will be useful for identifi cation of biomarkers for cholestasis and for determination of adaptive molecular mechanisms in cholestasis.