Cyp2c70 is responsible for the species difference in bile acid metabolism between mice and humans

CYP, cytochrome P450; -cluster null; Cyp2c -null, Cyp2c -cluster null; Cyp2d -null, Cyp2d cluster null; Cyp3a -null, Cyp3a -cluster null; CYP7a1, cholesterol 7 α -hydroxylase; FGF15, fibroblast growth factor 15; FXR, farnesoid X receptor; HBSS, Hank's buffered salt solution; h CYP2C9 , CYP2C9 humanized; HDCA, hyodeoxycholic acid; LCA, lithocholic acid; α -MCA, α -muricholic acid; β -MCA, β muricholic acid; PBS, phosphate-buffered saline; Ppia , peptidylprolyl isomerase A; T- α -MCA, tauro- α muricholic acid ; T- β -MCA, tauro- β -muricholic acid; ω -MCA, ω -muricholic acid; T-CDCA, tauro-chenodeoxycholic acid; T-LCA, tauro-lithocholic acid; Abstract Bile acids are synthesized from cholesterol in the liver and subjected to multiple metabolic biotransformations in hepatocytes, including oxidation by cytochromes P450 (CYP)s and conjugation with taurine, glycine, glucuronic acid, and sulfate. Mice and rats can hydroxylate chenodeoxycholic acid (CDCA) at the 6 β -position to form α -muricholic acid ( α -MCA), and ursodeoxycholic acid (UDCA) to form β -muricholic acid ( β -MCA). However, MCA is not formed in humans to any appreciable degree and the mechanism for this species difference is not known. Comparison of several Cyp -null mouse lines revealed that α -MCA and β -MCA were not detected in the liver samples from Cyp2c -cluster null ( Cyp2c -null) mice. Global bile acids analysis further revealed the absence of MCA and their conjugated-derivatives, and high concentration of CDCA, UDCA in Cyp2c- null mouse cecum and feces. Analysis of recombinant CYPs revealed that α -MCA and β -MCA were produced by oxidation of CDCA and UDCA by Cyp2c70. CYP2C9 -humanized mice have similar bile acid metabolites as the Cyp2c -null mice, indicating that human CYP2C9 does not oxidize CDCA and UDCA thus explaining the species differences in production of MCA. Since humans do not produce MCA, they lack tauro- β -MCA, a farnesoid X receptor (FXR) antagonists in mouse, that modulates obesity, insulin resistance and hepatosteatosis.


Introduction
Bile acids are synthesized from cholesterol in the liver and secreted through the biliary tract into the small intestine, where they aid in the absorption of lipids and fat-soluble vitamins (1,2). Greater than 90% of bile acids produced in the liver are reabsorbed in the small intestine in the process of enterohepatic circulation. Bile acid synthesis and transport in the liver and intestine is regulated by the farnesoid X receptor (FXR, NR1H4), a member of the nuclear receptor superfamily (3,4). Bile acids are subject to multiple metabolic biotransformations in hepatocytes, including conjugation with taurine, glycine, glucuronic acid, and sulfate (5). Mice and rats can hydroxylate chenodeoxycholic acid (CDCA) at the 6β-position to form α-muricholic acid (α-MCA), and ursodeoxycholic acid (UDCA) to form βmuricholic acid (β-MCA). MCA is produced in both mouse and rat liver, but is not formed at significant levels in human liver, thus indicating a species difference in MCA synthesis. In addition, mice mainly produce taurine conjugates of bile acids, while humans produce both mostly glycine conjugates and some taurine conjugates (6); rats also carry out both taurine and glycine conjugation. This is of particular interest since the taurine conjugate of β-MCA, T-β-MCA, is an antagonist of FXR in the ileum that controls FXR signaling and metabolic disease in mouse models of obesity (7)(8)(9).
While the hepatic cytochromes P450 (CYP) play a central role in the metabolism of drugs, toxins, and carcinogens, they also carry out key metabolic reactions in steroid hormone and bile acid synthesis. A number of CYPs participate in the synthesis of bile acid metabolites, with cholesterol 7α-hydroxylase (CYP7A1) generally considered the rate limiting enzyme in bile acid synthesis (10). Cyp7a1 is under control of FXR in the liver, and by FXR in the intestine through modulation of fibroblast growth factor 15 (FGF15) produced in the intestine that suppresses Cyp7a1 expression in the liver (11). The mammalian CYPs have remarkable diversity between species, with 57 CYP genes identified in humans, and more than 4 specificity, and multiplicity of CYPs between species, significant efforts have been made to develop and characterize Cyp-null and CYP-humanized mice, with the aim to determine the metabolic functions of CYPs and to provide mouse models that better predict human pathways of metabolism (14,15). Viable knockout models of the mouse Cyp3a (16,17), Cyp2d (18), Cyp1a (19) and Cyp2c (20) gene clusters were described, which are associated with metabolic differences in the metabolism of xenobiotics as compared with wild-type controls.
In present study, Cyp-null mice were used to determine which CYPs are responsible for the differences in production of bile acid metabolites between humans and mice, notably the hepatic synthesis of MCA. Individual BA concentrations were determined in wild-type, Cyp-null mice leading to the identification of Cyp2c70 as the CYP responsible for MCA synthesis in mice. This was confirmed by recombinant Cyp2c70 expression and Cyp2c70 siRNA inhibition in primary mouse hepatocytes.

Animal maintenance and treatments
All animal studies and procedure were carried out in accordance with Institute of Laboratory Animal Resources guidelines and approved by the National Cancer Institute Animal Care and Use Committee.
Mice were housed in a pathogen-free animal facility under a standard 12-hour light/dark cycle and given pelleted NIH-31 chow diet and water ad libitum. Male mice between 8 and 12 weeks of age were used for isolation of primary hepatocytes and preparation of liver microsomes. Liver tissue, fecal samples and ileum/cecum contents for metabolomics analysis were collected in Dundee and shipped to the NCI for analysis. Adult Cyp1a-cluster null (Cyp1a-null) (19), Cyp2c-cluster null (Cyp2c-null) (20), Cyp2d-cluster null (Cyp2d-null)(18), Cyp3a-cluster null (Cyp3a-null) (16) and CYP2C9-humanized (hCYP2C9) (20) mice were housed singly in open-top cages with ad libitum 24 hours access to food and water. RM1A chow diet (Special Diets Services, Witham, UK) was removed for the final 4 hours before killing. After 5 killed by CO 2 asphyxiation and blood collected by cardiac puncture. Tissues were excised and immediately frozen in liquid nitrogen and serum and tissues were stored at -80°C until use.

Measurement of mRNAs
Total RNA of liver was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA). qPCR was performed using cDNA generated from 1 µg total RNA with qScript™ cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD). qPCR reactions were carried out using SYBR green qPCR master mix (Biotools, Houston, TX) in an QuanStudio TM 7 Flex System. The primer pairs were designed using Primer-BLAST (National Center for Biotechnology Information), and was shown in Supplementary

Quantification of bile acid metabolites
Twenty mg of liver, cecum and feces were homogenized with 200 µL of 100% acetonitrile containing 1 µM d5-taurocholate (Sigma-Aldrich) as an internal standard and centrifuged twice at 15,000 × g for 25 minutes at 4°C for removal of precipitated proteins and other particulates. The supernatant was diluted by an equal volume of HPLC grade water (Thermo Fisher Scientific, Waltham, MA) containing 0.1% formic acid. Quantification of bile acid metabolites were measured as described previously (7,9). LC-MS was performed on a Waters Acquity H-Class UPLC system using a Waters Acquity BEH C18 column (2.1 × 100 mm) coupled to a Waters Xevo G2 QTOFMS. UPLC was performed by the following protocol: A, 0.1% Formic acid in water and B, 0.1% formic acid in acetonitrile. An initial gradient of 80% A for 4 minutes, to 60% A at 15 minutes, to 40% A at 20 minutes, to 10% A at 21 minutes, followed by flushing

Preparation and treatment of mouse primary hepatocytes.
Primary hepatocytes were isolated from C57BL/6J mice as described previously (21). Briefly, after killing mice by CO 2 asphyxiation, the abdomen was incised and mesentery and intestine moved to expose the portal vein. A cannula was inserted into the portal vein and liver was perfused with 40 ml of Hank's buffered salt solution (HBSS) without magnesium or calcium (Thermo Fisher Scientific) containing 1 mM EDTA at 4 ml/min. Blood was extravasated by cutting the inferior vena cava. After perfusion of the entire liver using 50 ml of HBSS containing collagenase I and II (0.6 mg/ml each, Thermo Fisher Scientific) and calcium chloride dehydrate (5 mM) at the speed of 4 ml/min, the digested liver was removed and placed in a sterile 10-cm Petri dish with 10 mM phosphate-buffered saline (PBS). The hepatic capsule was torn by fine-tip forceps and dispersed cells were filtered through 70-µm cell strainer (Becton Dickinson and Company) into a 50-ml tube and centrifuged at 200 × g, at 4 °C for 2 minutes.
Hepatocytes were further washed and purified by gradient centrifugation using Percoll Plus (GE Healthcare, Buckinghamshire, UK). After washing with HBSS and trypan blue staining, the number of hepatocytes were counted and then seeded in collagen-coated 12-well plates (Becton Dickinson and Company) at a density of 4 × 10 5 cells/well. Primary hepatocytes were cultured in William's E medium (Thermo Fisher Scientific) with 10% fetal bovine serum, and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Four-to six hours after seeding, the cells were treated with 10 nM Cyp2c29 Silencer ® Select Pre-designed siRNA (Thermo Fisher Scientific), Cyp2c70 Silencer ® Select Pre-designed siRNA (Thermo Fisher Scientific), and Silencer ® Select Negative Control #1 (Thermo Fisher Scientific) using Lipofectamine ® RNAiMAX (Thermo Fisher Scientific). Twenty-four hours after siRNA treatment, the cells were treated with medium containing CDCA-d4 or UDCA-d4 for 4 hours. At the prescribed time 8 points, medium and cells were harvested and subjected to measurement bile acids using QTOF-MS and qPCR respectively.

Statistical Analysis.
Statistical analysis was performed with Prism version 6 (GraphPad Software). Appropriate statistical analysis was applied, assuming a normal sample distribution. When comparing two groups, statistical significance was determined using two-tailed Student's t-test. When more than two groups were investigated, one-way ANOVA followed by Tukey's post-hoc correction was applied for comparisons. A p value of less than 0.05 was considered as significant difference. Results are expressed as the mean value and SD values.

MCA and conjugated-MCA in the liver of Cyp-null mice
To determine the influence of CYPs on the formation of α-MCA and β-MCA, bile acids were measured in livers of Cyp1a-null, Cyp2c-null, Cyp2d-null and Cyp3a-null mice. α-MCA and β-MCA were not detected in livers from Cyp2c-null mice, although they were present in other Cyp-null mouse lines ( Figure 1A). Moreover, T-α-MCA and T-β-MCA were also not detected in Cyp2c-null mouse livers ( Figure 1B). These results suggest that mouse Cyp2c might produce α-MCA and β-MCA.

Kinetic analysis of MCA production in vitro
To investigate MCA production from CDCA and UDCA, the activities of α-MCA and β-MCA production were measured using liver S9 fractions of wild-type, Cyp2c-null, and Cyp3a-null mice at 50 µM CDCA-d4 or 500 µM UDCA-d4 (Figure 2A, 2B). α-MCA-d4 was detected in CDCA-d4-treated S9 from wild-type and Cyp3a-null mice, but was not detected in S9 from Cyp2c-null mice (Figure 2A). β-MCA-d4 was not detected in any of the CDCA-d4-treated groups. α-MCA-d4 was not detected in the 9 UDCA-d4 treated-group while β-MCA was detected in S9 from wild-type and Cyp3a-null mice but was undetected in Cyp2c-null mice ( Figure 2B). As a control, it was confirmed that liver S9 fractions from all of mouse lines showed the 7-ethoxyresorufin (7-ER) O-deethylase activity, which is a marker activity of mouse Cyp1a2 ( Figure 2C). Kinetic analyses using different concentrations of CDCA and UDCA were performed using liver microsomes to determine the Km and Vmax values for α-MCA and β-MCA productions from CDCA and UDCA, respectively. The Km and Vmax values for α-MCA production were 8.19 ± 0.24 µM and 0.58 ± 0.01 nmol/min/mg, and those for β-MCA production were 321 ± 46 µM and 0.418 ± 0.03 nmol/min/mg, respectively ( Figure 2D). These data indicate that the catalytic efficiency for α-MCA production is higher than that for β-MCA production.

Levels of individual Cyp2c mRNAs in wild-type mice
There are 16 kinds of Cyp2c genes in mice, including Cyp2c53 that is a pseudogene, and their proteins are mainly expressed in the liver. The expression of each Cyp2c mRNA in liver was determined by RT-qPCR. The levels of Cyp2c29, Cyp2c50, Cyp2c67, Cyp2c69, and Cyp2c70 mRNAs were 5.3-, 1.8-, 3.0-, 1.5-and 1.9-fold that of Cyp2c44 mRNA, which is the only Cyp2c gene not deleted in the Cyp2cnull mice (Figure 3). The mouse Cyp2c cluster, with the exception of Cyp2c44, which is located 4 Mb away from the main Cyp2c gene cluster, was flanked with Cre recombinase recognition (loxP) sites using two consecutive rounds of targeting in mouse ES cells (20).

CDCA and UDCA oxidation activity by recombinant mouse Cyp2c70
To investigate which mouse Cyp2c isoform produces α-MCA from CDCA, Cyp2c29 and Cyp2c70 were transiently expressed in HepG2 cells as revealed by mRNA expression ( Figure 4A). Recombinant Cyp2c29 and Cyp2c70 showed the production of α-MCA-d4 from CDCA was only detected in cells expressing Cyp2c70. β-MCA was not detected in these groups ( Figure 4B). In contrast, α-MCA-d4 was by guest, on September 26, 2016 www.jlr.org Downloaded from not detected when UDCA-d4 was used as a substrate. Cyp2c70-expressing cells also showed β-MCA production from UDCA ( Figure 4C). These results suggest that Cyp2c70 catalyzes CDCA and UDCA oxidations to α-MCA and β-MCA, respectively.

Knockdown of Cyp2c70 reduces MCA production in mouse primary hepatocytes
To further confirm whether Cyp2c70 produces MCA from CDCA or UDCA, siRNA against Cyp2c29 (Si-Cyp2c29) and Cyp2c70 (Si-Cyp2c70) were transfected to mouse primary hepatocytes. Si-Cyp2c29 and si-Cyp2c70 significantly decreased Cyp2c29 and Cyp2c70 mRNA levels, respectively, and did not affect Cyp2c44 mRNA expression ( Figure 5A). In the presence of CDCA, α-MCA levels were specifically decreased in the si-Cyp2c70-treated cells, while β-MCA production was lowered in mouse primary hepatocytes in the presence of UDCA. This result reveals that Cyp2c70 is responsible for MCA production from CDCA and UDCA in mouse liver.

Bile acids in the liver, cecum and feces from Cyp2c-null mice and CYP2C9-humanized mice
To determine the consequence of loss of Cyp2c70 in the Cyp2c-null and CYP2C9-humanized mice, bile acids were measured in liver, cecum and feces of wild-type, Cyp2c-null, and CYP2C9-humanized mice. hCYP2C9 mice were generated from the Cyp2c-deleted ES cells described above by further Cremediated insertion of an expression cassette in which the human CYP2C9 gene is under control of the liver-specific mouse albumin promoter (20). In this study, the CYP2C9-humanized mice were used for the human model, because among human CYP2C enzymes, CYP2C9 is most abundant in the liver and is involved in the metabolism of various kinds of endogenous and exogenous compounds. α-MCA and β-MCA and their taurine conjugates were not detected in the liver, cecum and feces of Cyp2c-null and CYP2C9-humanized mice ( Figure 6). Moreover, the contents of CDCA and UDCA, which are precursor substances of α-MCA and β-MCA in mice, respectively, in Cyp2c-null and CYP2C9-humanized mice were significantly higher than those in wild-type mice. The contents of T-CDCA and T-UDCA were also high in Cyp2c-null and CYP2C9-humanized mice. In cecum and feces, lithocholic acid (LCA) was also significantly highly detected in Cyp2c-null and CYP2C9-humanized mice as compared with wild-type mice. In addition, tauro-LCA (T-LCA) was highly detected in these mice.

DISCUSSION
An earlier study revealed that mice with hepatocyte-specific deletion of NADPH-cytochrome P450 reductase (22), which decreased all CYPs activities, were found to have marked differences in a bile acid compositions including lower MCA and taurine-conjugated MCA as compared to their wild-type counterparts (23). However, this study could not distinguish which CYP isoform produced MCA. The current work demonstrated that mouse Cyp2c70 is responsible for production of MCA from CDCA or UDCA. Cyp2c-null mice and CYP2C9-humanized mice did not produce any MCA, in contrast to wildtype mice and mouse lines lacking expression of Cyp1a, Cyp2d and Cyp3a isoform. Cyp2c-null mice also have high concentration of CDCA and UDCA (and their taurine conjugates), the substrates for α-MCA and β-MCA, respectively.
LCA is produced from CDCA in the human and mouse large intestine. LCA is present in only trace levels in mouse liver, likely due to the low concentrations of CDCA. LCA and T-LCA, which are present in very low levels in wild-type mice, were found at significant concentrations in Cyp2c-null and CYP2C9humanized mouse livers. The hepatic Cyp7a1 and Cyp8b1 mRNA levels were not significantly different between wild-type, Cyp2c-null and hCYP2C9 mice (Supplemental Fig. S3). The increased concentrations of CDCA in livers of Cyp2c-null mice led to the production of LCA through a 7-dehydroxylation reaction by gut bacteria (24). LCA is then reabsorbed to the liver where it is conjugated with taurine. This is of particular interest, since LCA is considered a toxic bile acid (25). Thus, the lack of MCA production indirectly causes an increase in LCA. However, LCA is efficiently conjugated in mice leading to decreased potential for hepatotoxicity. Indeed, the homozygous CYP2C9-humanized and Cyp2c-null mice appeared normal, could not be distinguished from wild-type mice, they had normal body weights, liver 12 weights, and fertility (Supplemental Fig. S4A). The Cyp2c-null mice show no evidence for liver damage as compared to wild-type mice, although an increase in ALT and AST was observed in some mice due to inter-animal variability this change was not significant. The only significant phenotypic change in the CYP2C9-humanized mice was a decrease in alkaline phosphatase activity, while the Cyp2c-null mice exhibited a similar change and a small but significant decrease in plasma high-density lipoprotein and cholesterol (20).
T-β-MCA was identified as a potent FXR antagonist in mice (9,26). Inhibition of intestinal FXR resulted in the alleviation of metabolic disease including obesity, insulin resistance and hepatic steatosis (7)(8)(9). However, the question arises whether humans bile acids would have similar effects on FXR since humans do not produce MCA and thus T-β-MCA due to a lack of a CYP2C enzyme activity similar to mouse Cyp2c70. It is also noteworthy that UDCA is elevated in the Cyp2c-null mice and shows potential FXR antagonist activity in humans (27). FXR signaling in mice is dependent on the relative local concentration of endogenous agonist and antagonist that result from bile acid metabolism in the liver and intestine. Body weights and hepatic lipid concentrations (triglyceride, total cholesterol, phospholipid and non-esterified fatty acid) were not significant changed in wild-type, Cyp2c-null and hCYP2C9 mice (Supplemental Fig. S4B). The impact of these changes on the susceptibility to metabolic disease in the Cyp2c-null mice is an area of great interest and will require analysis of high-fat diet-induced obesity, insulin resistance and fatty livers (7,9).
The mouse and the human CYP2C cluster differ significantly in their genomic organization, with 15 functional genes described in mice compared with only four genes in humans. Analysis of hepatic mRNAs revealed that Cyp2c29 is the most abundant while Cyp2c50, Cyp2c67, Cyp2c69, and Cyp2c70 mRNAs were expressed at similar levels (12). Humans do not have an obvious homolog of Cyp2c70, at least at the level of primary amino acid sequence comparison (CYP2C8. 76%; CYP2C9, 79%; CYP2C18, 79%; CYP2C19, 79%).However, there is significant protein homology between Cyp2c70 and CYP2C22 which is the rat homolog of Cyp2c70 (Table 1). This is noteworthy because rats also have MCA similar to 13 mice (28). Murine Cyp2c37/38/39 and Cyp2c40 genes are endogenous female specific Cyp2c genes.
However, a much smaller gender difference in expression characterized Cyp2c29 and Cyp2c70 when comparing wild-type males and wild-type females (29). These findings support the view that Cyp2c70 is a primary enzyme responsible for MCA production. No other specific substrates for Cyp2c70 have been reported.
In mice, β-MCA and T-β-MCA appear to be more abundant than α-MCA and T-α-MCA. However, kinetic studies revealed a higher affinity of Cyp2c70 for CDCA than UDCA. These results suggest that α-MCA production is higher than that of β-MCA due to increased catalytic efficiency toward CDCA in mouse. The microbiota is involved in the metabolism of bile acids, particularly dehydroxylation and deconjugation reactions (9,30,31). Epimerization from α-MCA to β-MCA might occur in mouse intestine by microbiome metabolism through enzymes other than CYPs. Oxidation and epimerization of the 7-hydroxy groups of bile acids in the intestine are carried out by hydroxysteroid dehydrogenase expressed by intestinal bacteria (32).
Some studies have investigated whether bile acid concentrations might be useful to differentiate among various liver diseases (33)(34)(35). Bile acids also act agonists or antagonists for nuclear receptor such as FXR, pregnane X receptor (NR1I2), vitamin D receptor (NR1I1) and the G protein-coupled bile acid receptor, TGR5 (36). However, it is known that bile salt composition markedly differs between various species (37). As noted above, T-β-MCA is an FXR antagonist in mouse intestine, but humans do not produce MCA (26).
In addition, humans make glycine conjugates of bile acids while mice only make taurine conjugates.
Thus, mice are a poor model to investigate and predict the influence of bile acid metabolites in human disease. Mice lacking expression of the Cyp2c cluster showed similar bile acids profiling to humans, but still made taurine conjugates. Perhaps humanizing the bile acid conjugating enzymes bile acid-CoA ligase and bile acid-CoA:amino acid N-acyltransferase would make a more complete bile acid-humanized mouse line. Further, the bile acid profiles indicate that the gut microbiota of the Cyp2c-null mice may not 14 optimally hydrolyze the taurine conjugates of CDCA and LCA since T-CDCA and T-LCA accumulate in the cecum and feces. Since mouse gut microbiota seldom encounter these conjugates, the Cyp2c-null mice may not be an accurate model for human bile acid metabolism. Therefore, a mouse model that is optimal to study human diseases related to bile acids may have to be colonized with human gut microbiota. Taken together, the present study revealed that Cyp2c70 is the principal enzyme involved in MCA production and is responsible for the differences in bile acid metabolite profile between humans and mice.