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Originally published In Press as doi:10.1074/jbc.M310258200 on December 17, 2003

J. Biol. Chem., Vol. 279, Issue 12, 11336-11343, March 19, 2004
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Feed-forward Regulation of Bile Acid Detoxification by CYP3A4

STUDIES IN HUMANIZED TRANSGENIC MICE*

Catherine Stedman{ddagger}§, Graham Robertson¶, Sally Coulter{ddagger}, and Christopher Liddle{ddagger}||**

From the Departments of {ddagger}Clinical Pharmacology and Medicine, University of Sydney, Molecular Pharmacology Laboratory and Storr Liver Unit, Westmead Millennium Institute and ||Institute of Clinical Pathology and Medical Research, Westmead Hospital, New South Wales 2145, Australia

Received for publication, September 16, 2003 , and in revised form, December 17, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bile acids are potentially toxic end products of cholesterol metabolism and their concentrations must be tightly regulated. Homeostasis is maintained by both feed-forward regulation and feedback regulation. We used humanized transgenic mice incorporating 13 kb of the 5' regulatory flanking sequence of CYP3A4 linked to a lacZ reporter gene to explore the in vivo relationship between bile acids and physiological adaptive CYP3A gene regulation in acute cholestasis after bile duct ligation (BDL). Male transgenic mice were subjected to BDL or sham surgery prior to sacrifice on days 3, 6, and 10, and others were injected with intraperitoneal lithocholic acid (LCA) or vehicle alone. BDL resulted in marked hepatic activation of the CYP3A4/lacZ transgene in pericentral hepatocytes, with an 80-fold increase in transgene activation by day 10. Individual bile acids were quantified by liquid chromatography/mass spectrometry. Serum 6{beta}-hydroxylated bile acids were increased following BDL, confirming the physiological relevance of endogenous Cyp3a induction to bile acid detoxification. Although concentrations of conjugated primary bile acids increased after BDL, there was no increase in LCA, a putative PXR ligand, indicating that this cannot be the only endogenous bile acid mediating this protective response. Moreover, in LCA-treated animals, 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside staining showed hepatic activation of the CYP3A4 transgene only on the liver capsular surface, and minimal parenchymal induction, despite significant liver injury. This study demonstrates that CYP3A up-regulation is a significant in vivo adaptive response to cholestasis. However, this up-regulation is not dependent on increases in circulating LCA and the role of other bile acids as regulatory molecules requires further exploration.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bile acids are physiological detergents that facilitate absorption, transport, and distribution of lipid-soluble fats and vitamins, and excretion of lipids and cholesterol. However, hydrophobic bile acids are inherently cytotoxic and their formation and elimination must be tightly regulated. This homeostasis is provided by two general pathways: (i) feed-forward regulation, where toxic bile acids trigger an adaptive protective response, which results in an increase in expression of a range of genes responsible for bile acid detoxification and/or excretion; and (ii) feedback regulation, where accumulating bile acids cause reduced transcription of genes such as cholesterol 7{alpha}-hydroxylase (CYP7A1),1 the rate-limiting step in bile acid formation by the "classic" pathway of bile acid synthesis (1). Whereas the mechanisms of feedback regulation are now relatively well understood, the processes involved in feed-forward regulation are less clear.

CYP3A enzymes are predominantly expressed in the liver and small intestinal mucosa and catalyze the metabolic conversion of a wide diversity of xenobiotics and endogenous substrates to more polar derivatives. These enzymes exhibit adaptive transcriptional regulation in response to a range of xenobiotic inducing chemicals, which include some therapeutic drugs. Cytochrome P450 3A4 (CYP3A4) is active in the hydroxylation of bile acids and steroid hormones (2, 3). This is significant because the relative cytotoxicity of each bile acid is attributable to its hydrophobicity, and both ring hydroxylations and conjugation reduce hydrophobicity and effectively detoxify bile acids as well as rendering them accessible to excretory transporters (4). Bile acid-mediated induction of CYP3A enzymes therefore represents an important potential feed-forward mechanism for bile acid homeostasis.

There is indirect evidence that CYP3A induction occurs in response to cholestasis. Increased excretion of the 6{alpha}-hydroxylated bile acids, {alpha}-hyocholic acid ({alpha}-HCA) and hyodeoxycholic acid (HDCA), occurs in cholestatic patients and in women with cholestasis of pregnancy (5–7). In rats, concentrations of the 6{beta}-hydroxylated bile acid, {beta}-muricholic acid ({beta}-MCA) increase after bile duct ligation (BDL) (8, 9). Moreover, whereas hepatic CYP concentrations are generally decreased in human livers with biliary cirrhosis secondary to chronic cholestatic liver diseases, CYP3A4 expression is relatively preserved (10), suggesting that CYP3A induction occurs as a defensive response to bile acid accumulation, even in advanced disease.

Adaptive regulation of CYP3A4 is now known to be mediated by members of the nuclear receptor superfamily that function as ligand-activated transcription factors. Specifically, the pregnane X receptor (PXR; NR1I2) (3, 11–14), constitutive androstane receptor (CAR; NR1I3) (15), and 1,25-dihydroxyvitamin D receptor (VDR; NR1I1) (16) have been implicated as important "sensors" for potentially toxic compounds that can impact on CYP3A expression. These receptors heterodimerize with the receptor for 9-cis-retinoic acid-{alpha} (NR2B1) and recognize a range of small lipophilic molecules that includes endobiotic (endogenously synthesized) and xenobiotic compounds.

Recently, PXR has been shown to be a low affinity receptor for lithocholic acid (LCA) and its metabolite, 3-keto-lithocholic acid (3-keto-LCA) (3, 11, 17). In studies of knockout and transgenic animals, activation of PXR can both induce CYP3A enzymes and confer resistance to toxicity by LCA (3, 11). It has therefore been suggested that LCA represents the endogenous PXR ligand, and that PXR-mediated gene induction may be the predominant pathway of feed-forward regulation of bile acid detoxification and elimination (3, 11, 18). However, concentrations of LCA and other bile acids have not been measured in conjunction with an assessment of CYP3A induction in in vivo models of cholestasis.

In the present study, we have utilized a recently described transgenic mouse model incorporating a regulatory human CYP3A4 transgene (19) to explore the in vivo relationship between specific bile acids and physiological adaptive CYP3A gene regulation in acute cholestasis induced by surgical BDL. We provide evidence that adaptive up-regulation of both endogenous mouse and introduced human CYP3A genes does occur in response to bile acid accumulation, and that this response is not dependent on increases in circulating LCA.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents—Authentic bile acid standards were purchased from Sigma, except glycolithocholic acid (GLCA), murideoxycholic acid (MDCA), HDCA, {alpha}-HCA, {beta}-MCA, {alpha}-MCA, and tauro-{beta}-muricholic acid (T{beta}-MCA) purchased from Steraloids (Newport, RI), taurocholic acid (TCA) from Calbiochem (San Diego, CA), and the deuterated bile acid standards cholic-2,2,4,4-d4 acid, chenodeoxycholic-2,2,4,4-d4 acid, and lithocholic-2,2,4,4-d4 acid from C/D/N Isotopes (Quebec, Canada). High performance liquid chromatography grade methanol was purchased from APS Chemicals (Sydney, Australia).

Animals and Operative Procedures—CYP3A4/lacZ transgenic mice carrying a construct comprising the upstream regulatory region of the human CYP3A4 gene, -13 kb to +53 bp relative to the transcription initiation site, linked to the lacZ reporter gene have been recently described in detail (19). Eight to 10-week-old line 9/4 male mice hemizygous for a -13CYP3A4/lacZ transgene were used for all experiments. The animals were kept in individual cages, fed on a commercial pellet diet and allowed water ad libitium. All animal protocols and studies performed were approved by the Western Sydney Area Health Service Animal Ethics Committee.

Bile Duct Ligation—Mice were anesthetized (ketamine 100 mg/kg, and xylazine 20 mg/kg, administered intraperitoneally) and aseptically subjected to double ligation of the common bile duct below the bifurcation and single ligation above the pancreas with dissection between the distal and proximal ligatures. Sham operations were performed by gently touching the common bile duct with forceps, instead of ligation and subsequent dissection. Animals (n = 4–5 per group at each time point) were sacrificed by exsanguination under anesthesia 3, 6, and 10 days after BDL or sham operation. Mortality and the frequency of complications was less than 20%. At the time of sacrifice, blood samples were collected for determination of alanine aminotransferase (ALT) and bilirubin as a measure of liver injury, and cholestasis, respectively. Serum ALT and bilirubin were analyzed by the Department of Clinical Chemistry, Institute for Clinical Pathology and Medical Research, Western Sydney Area Health Service, using automated procedures. Serum and urine samples were also frozen at -70 °C for subsequent bile acid analysis. Liver and duodenal tissues were collected for staining with 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal), and liver was snap frozen in liquid nitrogen for quantitative {beta}-galactosidase assessment, protein analysis, mRNA extraction, and bile acid analysis. Liver samples were also embedded in OCT compound for {beta}-galactosidase staining of frozen sections.

Bile Acid Administration—Mice were administered lithocholic acid, 0.125 mg/g body weight in 100 µl of corn oil or vehicle alone twice daily by intraperitoneal injection for 7 days. Mice were sacrificed by exsanguination 12 h after the last injection.

Assessment of Liver Histology—Consecutive sections of liver (4 µm thick) from paraffin-embedded liver were cut for hematoxylin and eosin staining for evaluation of liver injury and necrosis.

Analysis of CYP3A4 Transgene Expression—{beta}-Galactosidase activity was visualized in slices and frozen sections of liver and other tissues following staining with X-gal. Tissues were fixed in 0.25% glutaraldehyde, 0.1 M phosphate buffer, pH 7.3, 5 mM EGTA, 2 mM MgCl2: washed in 0.1 M phosphate buffer, pH 7.3, 0.01% sodium deoxycholate, 0.025% Nonidet P-40, and 2 mM MgCl2; and stained by incubation at 37 °C in wash solution supplemented with 1 mg/ml X-gal, 5 mM potassium ferricyanide, and 5 mM potassium ferrocyanide. Tissue slices were examined under a stereomicroscope (magnification, x20) and tissue sections using a conventional microscope (magnification, x100). The extent of {beta}-galactosidase activity was quantitated in whole liver homogenates (100 mg of fresh tissue/ml of 0.25 M Tris-HCl, pH 7.3) using the o-nitrophenyl-{beta}-D-galactopyranoside (ONPG) assay as described previously (20). After appropriate dilution, the homogenate was incubated with {beta}-galactosidase assay reagent (0.1 M sodium phosphate buffer, pH 7.3, 1 mM MgCl2, 50 mM {beta}-mercaptoethanol, and 0.88 mg/ml ONPG) at 37 °C, quenched by the addition of 1 M Na2CO3, and the absorbance at 420 nm was determined. Protein concentration was quantified in the same whole liver homogenates using a commercially available kit (DC Protein Assay; Bio-Rad), and the units of {beta}-galactosidase activity are given as A420/mg of protein/min.

Endogenous Mouse Cyp3a11 Gene Expression—Endogenous mouse Cyp3a11 mRNA expression was determined by real-time reverse transcriptase-polymerase chain reaction. RNA was extracted from liver using a commercially available reagent (Trizol, Invitrogen). cDNA was synthesized in duplicate from 5 µg of total RNA using random hexamers and Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. An aliquot of each cDNA synthesis reaction (1 µl) was subjected to PCR amplification in duplicate using a PE Applied Biosystems (Foster City, CA) Prism 7700 real-time PCR platform. Primers and TaqMan® probe were as follows: forward primer, bases 112–133: 5'-TGCTCCTAGCAATCAGCTTGG-3'; reverse primer, bases 220–199: 5'-GTGCCTAAAAATGGCAGAGGTT-3'; and probe, bases 137–171: 5'-FAM-CCTCTACCGATATGGGACTCGTAAACATGAACTT-TAMRA-3'. The probe was designed to cross an intron-exon junction to avoid interference from genomic DNA. Additionally, primer and probe sequences were chosen to avoid detection of other Cyp3a subfamily members. Results were normalized against glyceraldehyde-3-phosphate dehydrogenase, determined using the following primers and TaqMan® probe: forward primer: 5'-GTCGTGGATCTGACGTGCC-3'; reverse primer: 5'-TGCCTGCTTCACCACCTTCT-3'; and probe: 5'-VIC-CCTGGAGAAACCTGCCAAGTATGATGACA-TAMRA-3'. Cycle parameters for all PCR were: 50 °C for 2 min then 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.

Quantitation of Bile Acids by High Performance Liquid Chromatography/Mass Spectrometry—Serum or urine (100 µl) were diluted with 100 µl of phosphate-buffered saline to which 41 ng of deuterated assay internal standard (cholic acid) was added. Solid phase extraction was carried out using Oasis HLB cartridges (Waters, Milford, MA) as per the manufacturer's directions. Typical recoveries of extracted bile acids exceeded 85%. Mouse liver samples (50–100 mg) were homogenized with a Polytron (Kinematica CH6010, Kriens/Luzern, Switzerland) at 80% full speed for 10 s in a solution of Tris buffer, pH 7.4. Homogenates were brought to a final concentration of 100 mg/ml. After the addition of the assay internal standard (cholic-2,2,4,4-d4 acid, 100 ng) the bile acids were extracted from the supernatant fraction (saturated with 0.75 g of ammonium sulfate) with dichloromethane-isopropyl alcohol (80:20; 2x 4 ml). Combined extracts were dried down with nitrogen and reconstituted in 150 µl of the chromatographic mobile phase. Extraction efficiency for this method was determined by spiking liver homogenates with deuterated bile acids covering a range of hydrophobicities, namely cholic acid, chenodeoxycholic acid, and lithocholic acid. Recovery for all three bile acids was in the range of 85–100%.

Chromatographic separations were carried out with a Waters 2695 pump equipped with an autoinjector. The analytes were separated on a Waters X-Terra MS C18 column (3.5 micron, 2.1 x 150 mm). The mobile phase consisted of solvent A (water), solvent B (methanol), and solvent C (100 mM ammonium acetate, pH 4.5) delivered as a gradient: 0–15 min for solvent B, 67%; 15–25 min for solvent B, 67–85%; and 25–35 min for solvent B, 85–67%, with 10% solvent C at a constant flow rate of 0.2 ml/min. The high performance liquid chromatography was coupled with a Waters ZQ quadruple mass detector via an electrospray ionization interface operating in the negative ion mode. Quantitative determination of bile acids was performed by time scheduled single ion recordings using [M - H]- ions. We determined that the following tune parameters are optimal for bile acid detection; capillary voltage 3 kV, cone voltage 40 V, extractor voltage 5 V, and RF lens 0.3 V. Source temperature was 100 °C and desolvation temperature was 300 °C. Desolvation gas flow was set at 350 liters/h and cone gas flow rate was 60 liter/h. The limit of quantitation was 40 nmol/liter for individual bile acids.

Statistical Analysis—Treatment groups were compared using factorial analysis of variance, and post-hoc analysis was performed using the Bonferroni/Dunn test for parametric data. These results are presented as mean ± S.D. Data from bile acid analysis were non-parametric, and treatment groups were compared using the Kruskal-Wallis test and Mann-Whitney test. These results are presented as median and interquartile range. All results are considered significant when p is less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Liver Weight and Assessment of Liver Injury and Cholestasis after BDL—CYP3A4/lacZ transgenic male mice were subjected to BDL or sham operation, and sacrificed after 3, 6, and 10 days. Liver weight progressively increased after BDL, with a 2-fold increase in BDL animals compared with shams at day 10 (mean 2.5 ± 0.3 g versus 1.2 ± 0.01 g; p < 0.001). Serum bilirubin and ALT were abnormal in BDL animals at all time points, confirming both cholestasis and liver injury. There was a progressive increase in serum ALT to a mean of 1,607 ± 394 units/liters at 10 days after BDL (Fig. 1A), and serum bilirubin peaked 6 days after BDL with a mean of 137 ± 100 µmol/liter (Fig. 1B). At all time points, ALT and bilirubin were significantly greater in BDL animals compared with shams (p < 0.001).



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  		FIG. 1.
Sequential changes in liver function tests after BDL and sham operation. A, serum ALT; and B, serum bilirubin. Data are presented as mean values ± S.D. Serum ALT and bilirubin are both significantly greater in BDL mice than sham mice at all time points (p < 0.0001).

 
CYP3A4 Transgene Expression in Whole Liver and Duodenum after BDL—The response of the CYP3A4 regulatory transgene to acute cholestasis was assessed by visualizing {beta}-galactosidase activity in slices and frozen sections of liver and duodenum by staining with X-gal (see "Experimental Procedures"). Significant hepatic induction of the CYP3A4/lacZ transgene was visualized by X-gal staining 6 and 10 days after BDL, compared with minimal constitutive expression in sham operated animals (Fig. 2A). Staining of frozen liver sections with X-gal demonstrated the distribution of transgene induction in pericentral (zone 3) hepatocytes (Fig. 3). A quantitatively and qualitatively similar pattern of transgene induction has previously been observed when this line of transgenic mice were treated with the classical xenobiotic Cyp3a inducing agents dexamethasone, pregnenolone 16{alpha}-carbonitrile, and phenobarbital (19). When intestinal epithelium was examined, no significant difference in transgene expression was seen between BDL and sham animals, although the transgene was inducible in the gut following dexamethasone administration (not shown). This lack of intestinal transgene induction after BDL may be because of the effect of bile acid diversion from the gut.



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  		FIG. 2.
Hepatic response of the CYP3A4/lacZ transgene to bile acid overload, visualized in whole liver wedges. A, male mice harboring the -13CYP3A4/lacZ transgene were subjected to BDL or sham operation, as described under "Experimental Procedures." Hepatocytes exhibiting transgene expression are visualized as the darkly stained areas on the cut surface of the liver after X-gal treatment. Marked induction is seen on days 6 and 10 after BDL, in contrast to the constitutive level of expression seen in the sham liver. B, LCA or corn oil vehicle alone was administered to animals by intraperitoneal injection for 7 days. In LCA-treated animals, significant activation of the transgene is seen only on the liver capsular surface, despite significant areas of hepatic necrosis and cavitation within the liver parenchyma, as indicated by the arrow.

 



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  		FIG. 3.
Hepatic expression of CYP3A4/lacZ transgene after BDL or LCA administration. Frozen liver sections were stained with a {beta}-galactosidase stain and eosin counterstain. In control animals only a few hepatocytes contain transgene-derived {beta}-galactosidase activity after X-gal staining (panel A). In contrast, the CYP3A4/lacZ transgene is significantly induced in pericentral (zone 3) hepatocytes by days 6 (panel B) and 10 (panel C) after BDL, visualized as the blue-stained areas. LCA administration induces significant cavitation and necrosis in the liver parenchyma (panel D) as indicated by the arrow, but the transgene is induced only on the liver capsular surface.

 
Quantitative Expression of CYP3A4/lacZ Transgene and Endogenous Mouse Cyp3a11 after BDL—CYP3A4/lacZ transgene expression was quantified by measuring {beta}-galactosidase activity in whole liver homogenates using the ONPG assay. Transgene expression increased significantly from day 6 in BDL animals compared with shams, to a mean 80-fold increase in induction on day 10 (Fig. 4A). On univariate analysis of variance, a statistically significant interaction was found between the effects of the procedure (BDL) and time. There was a linear increase in ONPG at a rate of 0.43/day (S.E. = 0.007, p < 0.001) in the BDL group. The sham group remained unchanged over time (slope = -0.002, S.E. = 0.008, p = 0.8).



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  		FIG. 4.
Comparison of quantitative expression of the CYP3A4/lacZ transgene with the endogenous mouse Cyp3a11 gene. A, hepatic transgene expression was assessed by determining {beta}-galactosidase activity in total liver lysates using the ONPG assay. The units of {beta}-galactosidase activity are given as A420/mg of protein/min and expressed as mean ± S.D., n = four to five animals per treatment. Transgene expression was significantly increased 6 and 10 days after BDL (p < 0.001). B, hepatic expression of the Cyp3a11 gene was examined in the same mice by real-time reverse transcriptase-polymerase chain reaction and normalized for glyceraldehyde-3-phosphate dehydrogenase mRNA expression. Significant differences were observed between BDL and sham on both days 6 (p < 0.001) and 10 (p < 0.01). The data are presented as -fold induction relative to sham and expressed as mean ± S.D., n = four to five animals per treatment.

 
Cyp3a11 is an endogenous mouse Cyp3a gene and is the likely homologue of CYP3A4, and relative expression was determined using real-time quantitative reverse transcriptase-PCR analysis. A significant increase in Cyp3a11 expression was demonstrated both 6 (3.7-fold) and 10 days after BDL (Fig. 4B), in keeping with the physiological relevance of the CYP3A4/lacZ transgene induction.

Changes to Serum Bile Acid Concentrations after BDL— Individual serum bile acid concentrations were determined in BDL and sham mice at different time points using LC/MS (Table I). Concentrations of {beta}-MCA, a 6{beta}-hydroxylated bile acid, and its tauro conjugate were massively elevated in BDL animals compared with sham animals at all three time points, and {alpha}-MCA was also elevated on day 3. Additionally, an ion consistent with taurine-conjugated unsaturated trihydroxybile acids (m/z 512), believed to correspond to tauro-{Delta}22-muricholate isomers (21) was also ~40-fold greater than that of unconjugated {beta}-MCA in BDL animals compared with shams (data not shown).


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  		TABLE I
Serum bile acid concentrations in BDL and sham mice Mice were subjected to BDL or sham operation, and serum was collected after 3, 6, and 10 days for determination of bile acid concentrations by LC/MS, as described under "Experimental Procedures." The data are shown as the median plus interquartile range from three to five mice per group, and expressed in nanomole liter. The following bile acids were below the limit of quantitation (40 nmol/liter) at all time points: CDCA, GCDCA, UDCA, GUDCA, MDCA, {alpha}-HCA, HDCA, LCA, GLCA, and TLCA.

 
The tauro conjugate of the major murine primary bile acid, CA, was significantly increased at all time points after BDL, although changes in serum concentration of unconjugated CA and glycocholic acid (GCA) did not reach statistical significance. This greater magnitude of increase in TCA is consistent with the known preference of rodents to amidate with taurine as the major metabolic pathway for conjugation (22). Likewise, serum concentrations of taurochenodeoxycholic acid (TCDCA) were substantially increased after BDL, but there was no significant increase in unconjugated chenodeoxycholic acid (CDCA) or glycochenodeoxycholic acid (GCDCA). As expected, concentrations of secondary bile acids including LCA, deoxycholic acid (DCA), and HDCA did not increase after BDL, reflecting the interruption of passage of bile acids into the gut and lack of exposure to intestinal bacteria.

Hepatic and Urine Bile Acid Concentrations after BDL—The bile acid concentrations in liver are presented in Table II, and concentrations in urine are presented in Table III. The pattern of changes in both liver and urine is generally similar to that in serum, reflecting the massive increase in tauro conjugates and {beta}-hydroxylated metabolites of the primary bile acids. In particular, significant increases in conjugates of CA, CDCA, UDCA, and {beta}-MCA, and increases in {alpha}-MCA were noted in urine and livers of BDL animals. In liver, the tauro conjugate of UDCA was also specifically identified and shown to be significantly increased after BDL. Hepatic concentrations of taurodeoxycholic acid (TDCA) decreased after BDL.


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  		TABLE II
Hepatic bile acid concentrations in BDL and sham mice Mice were subjected to BDL or sham operation, and liver tissue was collected after 3, 6, and 10 days for determination of bile acid concentrations by LC/MS, as described under "Experimental Procedures." The data are shown as the median plus interquartile range from three to five mice per group, and expressed in nanomole/g. The following bile acids were below the limit of quantitation (0.1 nmol/g liver) at all timepoints: GCDCA, GUDCA, {alpha}-HCA, HDCA, LCA, GLCA, and TLCA.

 


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  		TABLE III
Urine bile acid concentrations in BDL and sham mice Mice were subjected to BDL or sham operation, and urine was collected after 3, 6, and 10 days for determination of bile acid concentrations by LC/MS, as described under "Experimental Procedures." The data are shown as the median and interquartile range from two to five mice per group, and expressed in nmol/liter. The following bile acids were below the limit of quantitation (40 nmol/liter) at all time to points: CDCA, UDCA, HDCA, LCA, GLCA, and TLCA.

 
Lithocholic Acid Administration—The above data show that bile acid accumulation in acute obstructive cholestasis is associated with up-regulation of a regulatory human CYP3A4/lacZ transgene, induction of endogenous mouse Cyp3a11 gene expression, and an increase in 6{beta}-hydroxylated bile acids. This occurs in the absence of any increase in LCA, a putative PXR ligand. We therefore further explored the role of LCA in CYP3A induction by administering LCA to mice by intraperitoneal injection (see "Experimental Procedures"). Liver tests confirmed hepatic injury in the LCA-treated group of mice, with a mean ALT of 690 ± 585 units/liter compared with 40 ± 9 units/liter in the vehicle-treated group (p = .004); however, serum bilirubin did not differ significantly between the two groups (3.3 ± 1.5 versus 2.4 ± 0.8 µmol/liter (p = 0.24).

CYP3A4 Transgene and Cyp3a11 Expression after LCA Administration—The effect of LCA on transgene expression was assessed by visualizing {beta}-galactosidase activity in slices and frozen sections of liver stained with X-gal. Significant hepatic induction of the CYP3A4/lacZ transgene was visualized only on the capsular surface of the liver, with minimal induction seen within the liver parenchyma, despite the presence of significant LCA-induced hepatic injury and necrosis (Fig. 2B). Staining of frozen liver sections with X-gal confirmed this pattern of capsular transgene induction, related primarily to the site of maximal exposure to LCA (Fig. 3D). The lack of parenchymal transgene expression is unlikely to be secondary to the presence of liver injury, as no parenchymal induction was seen after a shorter duration of treatment with LCA (data not shown). When {beta}-galactosidase activity was quantified in liver lysates using the ONPG assay, a non-significant increase in transgene expression was seen in LCA-treated animals compared with vehicle-treated animals (A420: 0.041 ± 0.044 versus 0.002 ± 0.004 x 103/mg protein/min, p = 0.12). Wide variation in results was seen, probably secondary to the differential pattern of transgene expression on liver wedges. The relative hepatic expression of Cyp3a11 was modestly elevated, 2.2-fold, in LCA-treated animals compared with vehicle-treated animals, p = 0.036.

Serum Bile Acids after LCA Administration—The serum concentrations of TCA, TCDCA, LCA, DCA, and TDCA were significantly increased after LCA administration (Table IV). MDCA, a 6{beta}-hydroxylated metabolite of LCA was also increased; however, {alpha}-MCA and {beta}-MCA were not significantly elevated.


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  		TABLE IV
Serum bile acid concentrations after LCA administration Mice were injected intraperitoneally with LCA for 7 days, and serum bile acid concentrations were determined by LC/MS, as described under "Experimental Procedures." The data are shown as the median plus interquartile range from three to four mice per group, and expressed in nanomole/liter. The following bile acids were below the limit of quantitation (40 nmol/liter) in both groups: GCA, GCDCA, UDCA, GUDCA, and GLCA.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Using humanized transgenic mice and BDL as a model of acute cholestasis, we have confirmed that CYP3A up-regulation is a significant in vivo adaptive response to the accumulation of toxic bile acids. Furthermore, this is the first study to comprehensively characterize the changes in individual serum, liver, and urinary bile acids following BDL in mice. We have shown that CYP3A induction is not dependent on increased concentrations of LCA, a putative PXR ligand, as this hydrophobic secondary bile acid does not accumulate in this model.

Enzymes belonging to the CYP3A subfamily have been recognized as having a role in bile acid metabolism in several species. In human liver microsomes, CYP3A enzymes mediate 6{alpha}-, 7{alpha}-, and 6{beta}-hydroxylation of LCA (3), and 6{alpha}-hydroxylation of T-CDCA (7). CYP3As are also active catalysts of steroid hormone 6{beta}-hydroxylation (2), and there is a strong correlation between 6{alpha}-hydroxylation of LCA, CYP3A levels, and testosterone 6{beta}-hydroxylation (7). In hamsters, CYP3A10 encodes a lithocholic acid 6{beta}-hydroxylase (23). Additionally, in humans, CYP3A4 is the predominant enzyme responsible for 25- and 26-hydroxylations of 5{beta}-cholestane-3{alpha}7{alpha}12{alpha}-triol and 27-hydroxylation of 5{beta}-cholestane-3{alpha}7{alpha}12{alpha},25-tetrol in the classic bile acid biosynthetic pathway (24, 25). In mice, Cyp3a11 is responsible for these microsomal side chain hydroxylations (25).

In healthy adult humans, bile acid hydroxylation represents a relatively minor pathway for bile acid elimination, and 6{alpha}-hydroxylated bile acids are only found in small amounts in bile, serum, and urine (7). However, in the presence of cholestasis, urinary concentrations of {alpha}-HCA and HDCA, the 6{alpha}-hydroxylated metabolites of CDCA and LCA, increase (5, 7). 6{alpha}-Hydroxylation is followed by 6-O-glucuronidation and excretion, and this pathway is believed to be important for the detoxification of these hydrophobic bile acids (26). In the present study, concentrations of {alpha}-HCA and HDCA were not increased after BDL, but this is expected as rodents preferentially 6{beta}-hydroxylate bile acids (26–29). Additionally, in rats HDCA is predominantly a secondary bile acid, derived from {beta}-MCA through 7{beta}-dehydroxylation and 6{beta}-hydroxyepimerization (30).

We have now shown that the 6{beta}-hydroxylated bile acids, {alpha}-MCA, and especially {beta}-MCA increase significantly after BDL in mice. These 6{beta}-hydroxylated products of CDCA are relatively hydrophilic allowing subsequent excretion in the urine (31). Similar results have been found in BDL rats with increases in {beta}-MCA (8, 9, 32) and bile acid 6{beta}-hydroxylase activity (27, 29). The enzyme responsible for 6{beta}-hydroxylation of CDCA and LCA in mice has not been characterized; however, Cyp3a11 is known to be active in hepatic microsomal testosterone 6{beta}-hydroxylation (33), an activity that is widely accepted as a marker for the CYP3A subfamily enzymes, and CDCA 6{beta}-hydroxylation is inducible by dexamethasone, a known inducer of Cyp3a11 (34). It therefore seems likely that the hepatic Cyp3a11 induction that we have demonstrated mediates the increase in bile acid 6{beta}-hydroxylation. However, recent work has demonstrated an increase in Cyp3a11 in LCA-fed, FXR-null mice, associated with a decrease in LCA 6{beta}-hydroxylation, an increase in LCA 6{alpha}-hydroxylation and an increase in testosterone 6{beta}-hydroxylation (35). This suggests that in mice, several enzymes may mediate stereospecific hydroxylase reactions of different bile acid and steroid substrates. Interestingly, in the present study BDL was associated with significantly higher concentrations of {beta}-hydroxylated bile acids and greater Cyp3a11 induction than LCA administration. The increase in {beta}-MCA was seen from day 3 after BDL, prior to the significant increase in Cyp3a11 expression. This is consistent with previous work in rats suggesting that an additional pathway may contribute to 6{beta}-hydroxylation of bile acids in biliary obstruction (9). An important aspect of the present study is the ability to study the response of a regulatory human CYP3A4 transgene to cholestasis. Our work confirms the earlier indirect evidence from human studies suggesting that CYP3A up-regulation is an important early protective response to cholestasis, and is associated with an increase in bile acid hydroxylation.

Changes in individual serum and urine bile acids after BDL have not previously been described in mice, presumably because of difficulties in analyzing the small volume samples obtainable. Using a highly efficient and sensitive LC/MS-based method, we have found an increase in conjugated CA, a dominant primary bile acid in normal rodent bile. This is consistent with previous studies in the rat (32), although an early transient increase in unconjugated CA has also been observed (36). The more hydrophobic primary bile acid, CDCA, is usually of negligible quantitative importance in healthy rats, but has been reported to increase in serum (8, 37) and bile (36) after BDL, and we have demonstrated a significant increase in TC-DCA in serum, liver, and urine of mice following the BDL procedure. Additionally, we have demonstrated a significant increase in TUDCA after BDL.

In the present study there was no accumulation of LCA or its conjugates after BDL. This was expected in the BDL model of cholestasis, which interrupts the normal flow of bile acids into the gut, as LCA is a secondary bile acid formed by 7-dehydroxylation of CDCA by intestinal bacteria. This raises the question of the exact mechanism for CYP3A induction in cholestasis. Previous investigators have proposed that LCA is the endogenous ligand for PXR, inducing CYP3A gene transcription and thus providing greater capacity for hepatic bile acid detoxification (3, 11). This conclusion has been reached on the basis of cell culture-based experiments and cell-free ligand affinity assays (38), showing that mouse and human PXR are preferentially activated by LCA and its metabolite, 3-keto-LCA, but not by a range of conjugated bile acids (11). LCA is a known CYP3A substrate, and activation of PXR confers resistance to LCA-induced hepatotoxicity in mice pre-treated with the potent PXR ligand pregnenolone 16{alpha}-carbonitrile. However, PXR also increases transcription of a phase II conjugating enzyme, dehydroepiandrosterone sulfotransferase, which sulfates LCA and facilitates its elimination (39), and this mechanism, rather than CYP3A induction, may also account for some of the PXR-conferred protection from LCA toxicity.

We provide substantial evidence that CYP3A induction in both rodents and humans is an important part of the adaptive response of the liver to detoxify bile acids in acute cholestasis, even when there is no increase in circulating LCA. Indeed, animals injected with supraphysiological concentrations of LCA only had pericapsular hepatic induction of the transgene, corresponding to the area of maximal exposure to this bile acid, with minimal parenchymal induction despite serum LCA concentrations sufficiently high to induce hepatic necrosis well away from pericapsular liver. Therefore the role of other bile acids as regulatory molecules must be considered. Other potential bile acid ligands for PXR in human and mouse include CDCA and DCA (3, 17). CDCA is a relatively hydrophobic and toxic primary bile acid (40). TCDCA is a substrate of CYP3A4 for 6{alpha}-hydroxylation in humans (7), but is a substrate for {beta}-hydroxylation in rats (29), and increased significantly in concentration after BDL in the present study. However, it has recently been reported that PXR function is inhibited in the presence of CDCA or CA through the up-regulation of small heterodimer partner (NR1I0) (41), and TCDCA has also been shown to reduce CYP3A-associated monooxygenase activities in vivo in rats (42). DCA is a secondary bile acid, and concentrations of TDCA decrease significantly after BDL. FXR also acts as a bile acid "sensor," and is activated by both the primary bile acids, CDCA and CA, and the secondary bile acids LCA, DCA, and their conjugated metabolites (38). Although FXR has been shown to modestly activate a CYP3A4 reporter gene in the presence of CDCA and DCA, it is unlikely to be a positive regulator of CYP3a11 as FXR also increases small heterodimer partner expression, and Cyp3a11 protein and mRNA can be dramatically increased in the absence of FXR (17). UDCA and TUDCA can also activate PXR, and induce CYP3A protein and associated testosterone 6{beta}-hydroxylase activity in primary human hepatocytes, but have not been studied against murine PXR (17).

In addition to PXR and FXR, other relevant members of the nuclear receptor NR1 subfamily include VDR and CAR. VDR is expressed in hepatic nonparenchymal sinusoidal cell populations and biliary cells (43), and can be activated by LCA and its major metabolites: 3-keto-LCA, G-LCA, and 6-keto-LCA, inducing expression of CYP3A at lower concentrations than those required for activation of PXR. However, these secondary bile acids were not elevated in the present study, and primary bile acids including CDCA, CA, UDCA, and their conjugated metabolites are not effective ligands for VDR (16). It therefore seems unlikely that VDR mediates the CYP3A up-regulation observed in the present study.

The nuclear receptor CAR is activated by some of the same ligands as PXR; regulates a subset of common genes, e.g. CYP3A and CYP2B; and can signal through the same signaling pathways (15, 44). CAR is less promiscuous than PXR and displays a high basal level of activity that can be reduced by the binding of either naturally occurring androstanes or xenobiotics (45). Human and mouse CAR receptors are not significantly activated by a range of bile acids including CA, 6-keto-LCA, 12-keto-LCA, taurocholanic acid, 3,7-diketocholanic acid, or 7-keto-DCA methyl ester. Indeed, several bile acids including CA, 6-keto-LCA, and 7-keto-LCA are efficacious transrepressors of both mouse and human CAR (15). Although human CAR is not activated by CDCA (38), murine CAR has not been tested against CDCA or DCA. The effect of CDCA and its conjugates on CAR and CYP3A regulation therefore requires further exploration.

In summary, the present study has shown that adaptive up-regulation of important CYP3A genes occurs in acute cholestasis, in conjunction with an increase in hydroxylated bile acid production, as part of a coordinated protective response to detoxify bile acids. We have characterized changes in serum, liver, and urinary bile acids in mice after BDL, and provide evidence that CYP3A induction is not dependent on increases in circulating LCA. Gaining further insights into the mechanisms by which bile acids trigger feed-forward adaptation of detoxification pathways is an important goal as it will facilitate the development of rational therapies for use in cholestatic liver disease.


   FOOTNOTES
 
* This work was supported in part by project grants from the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of financial support from the Clinical Hepatology Trust Fund Westmead Hospital, a Westmead Millennium Foundation Initiating Grant, New Zealand Gastroenterology Society/Ferring Pharmaceuticals Research Fellowship, and a National Health and Medical Research Council of Australia Postgraduate Medical Research Scholarship. Back

** To whom correspondence should be addressed: Dept. of Clinical Pharmacology, Westmead Hospital, Darcy Rd., Westmead, New South Wales 2145, Australia. Tel.: 61-2-9845-6086; Fax: 61-2-9845-8351; E-mail: chris_liddle{at}wmi.usyd.edu.au.

1 The abbreviations used are: CYP7A1, cholesterol 7{alpha}-hydroxylase; {alpha}-HCA, {alpha}-hyocholic acid; {beta}-MCA, {beta}-muricholic acid; {alpha}-MCA, {alpha}-muricholic acid; 3-keto-LCA, 3-keto-lithocholic acid; ALT, alanine aminotransaminase; BDL, bile duct ligation; CA, cholic acid; CAR, constitutive androstane receptor; CDCA, chenodeoxycholic acid; CYP, cytochrome P450; CYP3A, cytochrome P450 3A; DCA, deoxycholic acid; FXR, farnesoid X receptor; GCA, glycocholic acid; GCDCA, glycochenodeoxycholic acid; GLCA, glycolithocholic acid; GUDCA, glycoursodeoxycholic acid; HDCA, hyodeoxycholic acid; LC/MS, liquid chromatography/mass spectrometry; LCA, lithocholic acid; MDCA, murideoxycholic acid; OH, hydroxylated; ONPG, o-nitrophenyl-{beta}-D-galactopyranoside; PXR, pregnane X receptor or steroid and xenobiotic receptor; T{beta}-MCA, tauro-{beta}-muricholic acid; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid; TLCA, taurolithocholic acid; TUDCA, tauroursodeoxycholic acid; UDCA, ursodeoxycholic acid; VDR, 1,25-dihydroxyvitamin D receptor; X-gal, 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside. Back


   ACKNOWLEDGMENTS
 
We thank Karen Byth for advice regarding statistical analysis, Jacqueline Field and Sandie Bierach for helpful advice and initial assistance with animal procedures, and Alan Hoffman for helpful discussion regarding bile acid metabolism in different species. Animals were sourced through the transgenics facility of the Westmead Millennium Institute.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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