Regulation of bile acid metabolism in mouse models with hydrophobic bile acid composition

The bile acid (BA) composition in mice is substantially different from that in humans. Chenodeoxycholic acid (CDCA) is an end product in the human liver; however, mouse Cyp2c70 metabolizes CDCA to hydrophilic muricholic acids (MCAs). Moreover, in humans, the gut microbiota converts the primary BAs, cholic acid and CDCA, into deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. In contrast, the mouse Cyp2a12 reverts this action and converts these secondary BAs to primary BAs. Here, we generated Cyp2a12 KO, Cyp2c70 KO, and Cyp2a12/Cyp2c70 double KO (DKO) mice using the CRISPR-Cas9 system to study the regulation of BA metabolism under hydrophobic BA composition. Cyp2a12 KO mice showed the accumulation of DCAs, whereas Cyp2c70 KO mice lacked MCAs and exhibited markedly increased hepatobiliary proportions of CDCA. In DKO mice, not only DCAs or CDCAs but also DCAs, CDCAs, and LCAs were all elevated. In Cyp2c70 KO and DKO mice, chronic liver inflammation was observed depending on the hepatic unconjugated CDCA concentrations. The BA pool was markedly reduced in Cyp2c70 KO and DKO mice, but the FXR was not activated. It was suggested that the cytokine/c-Jun N-terminal kinase signaling pathway and the pregnane X receptor-mediated pathway are the predominant mechanisms, preferred over the FXR/small heterodimer partner and FXR/fibroblast growth factor 15 pathways, for controlling BA synthesis under hydrophobic BA composition. From our results, we hypothesize that these KO mice can be novel and useful models for investigating the roles of hydrophobic BAs in various human diseases. The bile acid (BA) composition in mice is substantially different from that in humans. Chenodeoxycholic acid (CDCA) is an end product in the human liver; however, mouse Cyp2c70 metabolizes CDCA to hydrophilic muricholic acids (MCAs). Moreover, in humans, the gut microbiota converts the primary BAs, cholic acid and CDCA, into deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. In contrast, the mouse Cyp2a12 reverts this action and converts these secondary BAs to primary BAs. Here, we generated Cyp2a12 KO, Cyp2c70 KO, and Cyp2a12/Cyp2c70 double KO (DKO) mice using the CRISPR-Cas9 system to study the regulation of BA metabolism under hydrophobic BA composition. Cyp2a12 KO mice showed the accumulation of DCAs, whereas Cyp2c70 KO mice lacked MCAs and exhibited markedly increased hepatobiliary proportions of CDCA. In DKO mice, not only DCAs or CDCAs but also DCAs, CDCAs, and LCAs were all elevated. In Cyp2c70 KO and DKO mice, chronic liver inflammation was observed depending on the hepatic unconjugated CDCA concentrations. The BA pool was markedly reduced in Cyp2c70 KO and DKO mice, but the FXR was not activated. It was suggested that the cytokine/c-Jun N-terminal kinase signaling pathway and the pregnane X receptor-mediated pathway are the predominant mechanisms, preferred over the FXR/small heterodimer partner and FXR/fibroblast growth factor 15 pathways, for controlling BA synthesis under hydrophobic BA composition. From our results, we hypothesize that these KO mice can be novel and useful models for investigating the roles of hydrophobic BAs in various human diseases. A mouse is the most commonly used laboratory animal to extrapolate investigations regarding human metabolism. However, numerous differences have been reported between mice and humans. Bile acids (BAs), the end products of cholesterol catabolism, take part in the intestinal digestion and absorption and are recycled via the enterohepatic circulation. The BA composition is a primary indicator of the metabolic difference between mice and humans (1Thakare R. Alamoudi J.A. Gautam N. Rodrigues A.D. Alnouti Y. Species differences in bile acids I. Plasma and urine bile acid composition.J. Appl. Toxicol. 2018; 38: 1323-1335Crossref PubMed Scopus (49) Google Scholar). Certain BAs are ligands of nuclear and transmembrane G protein-coupled receptors and regulate lipid and carbohydrate metabolism, inflammation, fibrosis, and carcinogenesis (2Schaap F.G. Trauner M. Jansen P.L. Bile acid receptors as targets for drug development.Nat. Rev. Gastroenterol. Hepatol. 2014; 11: 55-67Crossref PubMed Scopus (466) Google Scholar). Therefore, the BA composition can be a crucial factor for creating relevant mouse models of human diseases (3Rudling M. Understanding mouse bile acid formation: Is it time to unwind why mice and rats make unique bile acids?.J. Lipid Res. 2016; 57: 2097-2098Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 4Fickert P. Wagner M. Biliary bile acids in hepatobiliary injury - what is the link?.J. Hepatol. 2017; 67: 619-631Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Although there are several differences between humans and mice with respect to BA metabolism (5Li J. Dawson P.A. Animal models to study bile acid metabolism.Biochim. Biophys. Acta Mol. Basis Dis. 2019; 1865: 895-911Crossref PubMed Scopus (86) Google Scholar), the following two reactions determine the characteristic phenotype associated with the BA composition in mice. First, most of chenodeoxycholic acid (CDCA), an end product in human liver, is further metabolized to muricholic acids (MCAs) by CDCA 6β-hydroxylase in the mouse and rat liver (Fig. 1A). CDCA is a cytotoxic BA (6Schölmerich J. Becher M.S. Schmidt K. Schubert R. Kremer B. Feldhaus S. Gerok W. Influence of hydroxylation and conjugation of bile salts on their membrane-damaging properties–studies on isolated hepatocytes and lipid membrane vesicles.Hepatology. 1984; 4: 661-666Crossref PubMed Scopus (255) Google Scholar, 7Kitani K. Kanai S. Sato Y. Ohta M. Tauro α-muricholate is as effective as tauro β-muricholate and tauroursodeoxycholate in preventing taurochenodeoxycholate-induced liver damage in the rat.Hepatology. 1994; 19: 1007-1012Crossref PubMed Scopus (18) Google Scholar) and is the most potent physiological agonist of the FXR (NR1H4) (8Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Identification of a nuclear receptor for bile acids.Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2145) Google Scholar, 9Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. et al.Bile acids: natural ligands for an orphan nuclear receptor.Science. 1999; 284: 1365-1368Crossref PubMed Scopus (1829) Google Scholar). In contrast, MCAs are cytoprotective (7Kitani K. Kanai S. Sato Y. Ohta M. Tauro α-muricholate is as effective as tauro β-muricholate and tauroursodeoxycholate in preventing taurochenodeoxycholate-induced liver damage in the rat.Hepatology. 1994; 19: 1007-1012Crossref PubMed Scopus (18) Google Scholar) and have antagonistic effects on FXR (10Sayin S.I. Wahlstrom A. Felin J. Jantti S. Marschall H.U. Bamberg K. Angelin B. Hyotylainen T. Oresic M. Backhed F. Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-β-muricholic acid, a naturally occurring FXR antagonist.Cell Metab. 2013; 17: 225-235Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar). Second, the primary BAs, cholic acid (CA) and CDCA, are 7α-dehydroxylated by intestinal bacteria and transformed into the secondary BAs, deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. In mice and rats, these secondary BAs are converted to primary BAs by the hepatic BA 7α-hydroxylase (Fig. 1A). Compared with primary BAs, DCA and LCA are more effective in activating the Takeda G protein-coupled receptor 5 (TGR5) (11Kawamata Y. Fujii R. Hosoya M. Harada M. Yoshida H. Miwa M. Fukusumi S. Habata Y. Itoh T. Shintani Y. et al.A G protein-coupled receptor responsive to bile acids.J. Biol. Chem. 2003; 278: 9435-9440Abstract Full Text Full Text PDF PubMed Scopus (1088) Google Scholar). They are also more cytotoxic (6Schölmerich J. Becher M.S. Schmidt K. Schubert R. Kremer B. Feldhaus S. Gerok W. Influence of hydroxylation and conjugation of bile salts on their membrane-damaging properties–studies on isolated hepatocytes and lipid membrane vesicles.Hepatology. 1984; 4: 661-666Crossref PubMed Scopus (255) Google Scholar) and promote carcinogenesis (12Yoshimoto S. Loo T.M. Atarashi K. Kanda H. Sato S. Oyadomari S. Iwakura Y. Oshima K. Morita H. Hattori M. et al.Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome.Nature. 2013; 499 ([Erratum. 2014. Nature. 506: 396.]): 97-101Crossref PubMed Scopus (1374) Google Scholar, 13Louis P. Hold G.L. Flint H.J. The gut microbiota, bacterial metabolites and colorectal cancer.Nat. Rev. Microbiol. 2014; 12: 661-672Crossref PubMed Scopus (1528) Google Scholar). However, the biliary proportion of these secondary BAs in mice is markedly low (less than 3%) (14Meir K. Kitsberg D. Alkalay I. Szafer F. Rosen H. Shpitzen S. Avi L.B. Staels B. Fievet C. Meiner V. et al.Human sterol 27-hydroxylase (CYP27) overexpressor transgenic mouse model. Evidence against 27-hydroxycholesterol as a critical regulator of cholesterol homeostasis.J. Biol. Chem. 2002; 277: 34036-34041Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) compared with that in humans (∼20–30%) (15Makino I. Nakagawa S. Changes in biliary lipid and biliary bile acid composition in patients after administration of ursodeoxycholic acid.J. Lipid Res. 1978; 19: 723-728Abstract Full Text PDF PubMed Google Scholar). The specific rodent genes responsible for the CDCA 6β-hydroxylation and BA 7α-rehydroxylation were not determined for a long time. However, Takahashi et al. (16Takahashi S. Fukami T. Masuo Y. Brocker C.N. Xie C. Krausz K.W. Wolf C.R. Henderson C.J. Gonzalez F.J. Cyp2c70 is responsible for the species difference in bile acid metabolism between mice and humans.J. Lipid Res. 2016; 57: 2130-2137Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) recently found that MCAs were not detected in liver samples of Cyp2c-cluster-null mice (17Scheer N. Kapelyukh Y. Chatham L. Rode A. Buechel S. Wolf C.R. Generation and characterization of novel cytochrome P450 Cyp2c gene cluster knockout and CYP2C9 humanized mouse lines.Mol. Pharmacol. 2012; 82: 1022-1029Crossref PubMed Scopus (41) Google Scholar). They, therefore, concluded that Cyp2c70 was necessary for the 6β-hydroxylation of CDCA in mice. However, the specific rodent genes responsible for hepatic BA 7α-rehydroxylation were still not determined until recently. An early study on the purification and characterization of rat liver taurodeoxycholic acid (TDCA) 7α-hydroxylase showed that BA 7α-rehydroxylation was catalyzed by a cytochrome P450 (CYP) enzyme (18Murakami K. Okuda K. Purification and characterization of taurodeoxycholate 7α-monooxygenase in rat liver.J. Biol. Chem. 1981; 256: 8658-8662Abstract Full Text PDF PubMed Google Scholar). Another study using liver-specific CYP oxidoreductase KO mice showed that biliary TDCA levels were markedly elevated after feeding CA (19Kunne C. Acco A. Hohenester S. Duijst S. de Waart D.R. Zamanbin A. Oude Elferink R.P. Defective bile salt biosynthesis and hydroxylation in mice with reduced cytochrome P450 activity.Hepatology. 2013; 57: 1509-1517Crossref PubMed Scopus (20) Google Scholar), suggesting that 7α-rehydroxylation of BAs is catalyzed by CYP enzyme(s). In this study, we first identified the mouse and rat genes encoding BA 7α-hydroxylase and CDCA 6β-hydroxylase through a new approach using orthology, tissue distribution, and sexual dimorphism data of CYP (20Nelson D.R. Gene nomenclature by default, or BLASTing to Babel.Hum. Genomics. 2005; 2: 196-201Crossref PubMed Scopus (11) Google Scholar, 21Renaud H.J. Cui J.Y. Khan M. Klaassen C.D. Tissue distribution and gender-divergent expression of 78 cytochrome P450 mRNAs in mice.Toxicol. Sci. 2011; 124: 261-277Crossref PubMed Scopus (130) Google Scholar). Then, we generated double KO (DKO) mice to examine BA metabolism. These mice showed BA composition just as we had expected, and the BA pool was markedly reduced. However, much to our surprise, FXR was not activated. αMCA, βMCA, ωMCA, tauro-αMCA, tauro-βMCA, and tauro-ωMCA were purchased from Steraloids (Newport, RI). Taurohyodeoxycholic acid (THDCA), taurolithocholic acid 3-sulfate (TLCA-3S), and LCA 3-sulfate (LCA-3S) were obtained from Cayman Chemical (Ann Arbor, MI). Pooled male mouse liver microsomes (CD1), pooled female mouse liver microsomes (CD1), pooled male rat liver microsomes (Sprague-Dawley), pooled female rat liver microsomes (Sprague-Dawley), and pooled human liver microsomes were purchased from BD Biosciences (Franklin Lakes, NJ), and male CD1 mouse kidney microsomes were purchased from Sekisui XenoTech (Kansas City, KS). Cyp2a12−/−Cyp2c70−/− DKO mice were generated using the CRISPR-Cas9 system by the Laboratory Animal Resource Center, University of Tsukuba (Ibaraki, Japan) and Charles River Laboratories Japan, Inc. (Kanagawa, Japan). Mice were kept under pathogen-free conditions and a regular 12 h light-dark cycle (light period: 0600–1800), with free access to standard chow and water. This project was approved by the Animal Experiment Committees of the University of Tsukuba, Charles River Laboratories Japan, and Tokyo Medical University. The oligos, Cyp2a12 intron 2 CRISPR F (5′-caccATAGTTAGGGGAAGCGACAT-3′) and Cyp2a12 intron 2 CRISPR R (5′-aaacATGTCGCTTCCCCTAACTAT-3′), Cyp2a12 intron 4 CRISPR F (5′-caccGTCTTACAATCCAGGCGAGG-3′) and Cyp2a12 intron 4 CRISPR R (5′-aaacCCTCGCCTGGATTGTAAGAC-3′), Cyp2c70 intron 1 CRISPR F (5′-caccAGATGATTATTAGTGTACAG-3′) and Cyp2c70 intron 1 CRISPR R (5′-aaacCTGTACACTAATAATCATCT-3′), and Cyp2c70 intron 2 CRISPR F (5′-caccTGGAACAGTGACAAGAGCGA-3′) and Cyp2c70 intron 2 CRISPR R (5′-aaacTCGCTCTTGTCACTGTTCCA-3′) were annealed and inserted into the BbsI restriction site of the pX330 vector (Addgene plasmid 42230). Constructed plasmids (circular) were designated pX330-Cyp2a12 intron 2, pX330-Cyp2a12 intron 4, pX330-Cyp2c70 intron 1, and pX330-Cyp2c70 intron 2. Pregnant mare serum gonadotropin and human chorionic gonadotropin were injected into female C57BL/6J mice at 48 h intervals, and the mice were mated with male C57BL/6J mice. Fertilized ova were collected from the oviducts, and 5 ng/μl each of pX330-Cyp2a12 intron 2 and pX330-Cyp2a12 intron 4 or pX330-Cyp2c70 intron 1 and pX330-Cyp2c70 intron 2 were injected into the pronuclei according to standard protocols (22Gordon J.W. Ruddle F.H. Integration and stable germ line transmission of genes injected into mouse pronuclei.Science. 1981; 214: 1244-1246Crossref PubMed Scopus (428) Google Scholar). The injected one-cell embryos were transferred into pseudopregnant CD1 mice. Using genomic DNA obtained from tail clippings, founder (F0) mice were selected by PCR followed by direct sequencing as described by Hoshino et al (23Hoshino Y. Mizuno S. Kato K. Mizuno-Iijima S. Tanimoto Y. Ishida M. Kajiwara N. Sakasai T. Miwa Y. Takahashi S. et al.Simple generation of hairless mice for in vivo imaging.Exp. Anim. 2017; 66: 437-445Crossref PubMed Scopus (8) Google Scholar). The sequences of the oligonucleotide primer pairs used were forward (F): 5′-GAGAGGCAAA-TGGGAACAAA-3′ and reverse (R): 5′-AACAGGCAGAAGCAGG-GATA-3′ for WT Cyp2a12, F: 5′-GAGAGGCAAATGGGAACAAA-3′ and R: 5′-AGGACCTCGGGATGAGAAGT-3′ for mutant Cyp2a12, F: 5′-TCTTCTTGCCTTCAACAGCA-3′ and R: 5′-AACCATTGCACAGAGCACAG-3′ for WT Cyp2c70, F: 5′-TCTTCTTGCCTTCAACAGCA-3′ and R: 5′-GAAAGCCCATGAGAGAGGAA-3′ for mutant Cyp2c70, and F: 5′-AGTTCATCAAGCCCATCCTG-3′ and R: 5′-GAAGTTTCTGTTGGCGAAGC-3′ for Cas9 detection. F0 mice for Cyp2a12-null and Cyp2c70-null were bred with WT C57BL/6J mice to determine their germline competency. A male F1 Cyp2a12+/− mouse was crossed with female C57BL/6J mice by in vitro fertilization using CARD HyperOva (Kyudo Co., Saga, Japan), and the resulting female Cyp2a12+/− mice were crossed with a male F1 Cyp2c70+/− mouse by in vitro fertilization using CARD HyperOva. Then, these double heterozygous Cyp2a12+/−Cyp2c70+/− animals were crossed to obtain Cyp2a12−/−Cyp2c70+/+ (2a12KO), Cyp2a12+/+Cyp2c70−/− (2c70KO), and Cyp2a12−/−Cyp2c70−/− (DKO) mice. All experiments reported here were performed with subsequent generations of these animals. Mice that were 10–12 weeks old [11.1 ± 0.8 (mean ± SD), n = 44] were used. After making them fast for 4 h with free access to water, they were euthanized between 1100 and 1600 under combination anesthesia with medetomidine, midazolam, and butorphanol. Their gallbladder, blood (serum), liver, small intestine, cecal contents, and feces were collected immediately and frozen at −80°C. Serum activities of alanine transaminase (ALT) and alkaline phosphatase (ALP) were determined by colorimetric assays using Transaminase CII-Test Wako and LabAssay ALP (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). BA concentrations were determined as described by Murakami et al. (24Murakami M. Iwamoto J. Honda A. Tsuji T. Tamamushi M. Ueda H. Monma T. Konishi N. Yara S. Hirayama T. et al.Detection of Gut Dysbiosis due to Reduced Clostridium Subcluster XIVa Using the Fecal or Serum Bile Acid Profile.Inflamm. Bowel Dis. 2018; 24: 1035-1044Crossref PubMed Scopus (29) Google Scholar) with minor modifications. Liver, small intestine, cecal contents, and feces were solubilized in 5% KOH/water at 80°C for 20 min; this heating step was omitted for serum and bile samples. After the addition of internal standards and 0.5 M potassium phosphate buffer (pH 7.4), BAs were extracted with Bond Elut C18 cartridges and quantified by LC-MS/MS. Chromatographic separation was performed using a Hypersil GOLD column (200 × 2.1 mm, 1.9 μm; Thermo Fisher Scientific) at 40°C. The mobile phase consisted of (A) 20 mM ammonium acetate buffer (pH 7.5)-acetonitrile-methanol (70:15:15, v/v/v) and (B) 20 mM ammonium acetate buffer (pH 7.5)-acetonitrile-methanol (30:35:35, v/v/v). The following gradient program was used at a flow rate of 150 μl/min: 0–50% B for 20 min, 50–100% B for 10 min, hold 100% B for 15 min, and re-equilibrate to 100% A for 10 min. Detailed LC-MS/MS conditions are presented in supplemental Table S1. Serum and hepatic concentrations of total cholesterol and triglycerides were measured by colorimetric assays using Cholesterol E-Test Wako and Triglyceride E-Test Wako (FUJIFILM Wako Pure Chemical Corporation), respectively. Biliary cholesterol and phospholipid concentrations were determined by Cholesterol E-Test Wako and Phospholipid C-Test Wako, respectively. Sterol and oxysterol concentrations in the liver and serum were quantified using our previously described LC-MS/MS method (25Honda A. Miyazaki T. Ikegami T. Iwamoto J. Yamashita K. Numazawa M. Matsuzaki Y. Highly sensitive and specific analysis of sterol profiles in biological samples by HPLC-ESI-MS/MS.J. Steroid Biochem. Mol. Biol. 2010; 121: 556-564Crossref PubMed Scopus (52) Google Scholar). Briefly, 5 μl of serum or 5 mg of liver tissue were incubated with internal standards in 1 N ethanolic KOH at 37°C for 1 h. Sterols were extracted with n-hexane, derivatized to picolinyl esters, and analyzed by LC-MS/MS. Microsomes and mitochondria were prepared from livers by differential ultracentrifugation (26Honda A. Salen G. Matsuzaki Y. Batta A.K. Xu G. Leitersdorf E. Tint G.S. Erickson S.K. Tanaka N. Shefer S. Differences in hepatic levels of intermediates in bile acid biosynthesis between Cyp27–/– mice and CTX.J. Lipid Res. 2001; 42: 291-300Abstract Full Text Full Text PDF PubMed Google Scholar). Microsomal activities of BA 6β-hydroxylase and BA 7α-hydroxylase were measured as follows: microsomes (100 μg of protein) were incubated for 20 min at 37°C with 200 μM of each BA (dissolved in 10 μl of 50% acetone in water), NADPH (1.2 mM), glucose-6-phosphate (3.6 mM), 1 unit of glucose-6-phosphate dehydrogenase, and 100 mM of potassium phosphate buffer (pH 7.4) containing 0.1 mM of EDTA in a total volume of 250 μl. The incubation was stopped by the addition of 10 μl of 8.9 M aqueous KOH solution. After the addition of internal standards and 0.5 M potassium phosphate buffer (pH 7.4), BAs were extracted with Bond Elut C18 cartridges and quantified by LC-MS/MS as described above. Instead of using microsomes, recombinant rat CYP2A1 (Supersome) prepared from insect cells (Corning, NY) was used to determine BA 7α-hydroxylation. Microsomal HMG-CoA reductase activity was measured by LC-MS/MS method, as described previously (27Honda A. Mizokami Y. Matsuzaki Y. Ikegami T. Doy M. Miyazaki H. Highly sensitive assay of HMG-CoA reductase activity by LC-ESI-MS/MS.J. Lipid Res. 2007; 48: 1212-1220Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The activities of microsomal cholesterol 7α-hydroxylase (CYP7A1) (28Honda A. Salen G. Shefer S. Batta A.K. Honda M. Xu G. Tint G.S. Matsuzaki Y. Shoda J. Tanaka N. Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7α-hydroxylase and 27-hydroxylase activities in rat liver.J. Lipid Res. 1999; 40: 1520-1528Abstract Full Text Full Text PDF PubMed Google Scholar), mitochondrial cholesterol 27-hydroxylase (CYP27A1) (28Honda A. Salen G. Shefer S. Batta A.K. Honda M. Xu G. Tint G.S. Matsuzaki Y. Shoda J. Tanaka N. Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7α-hydroxylase and 27-hydroxylase activities in rat liver.J. Lipid Res. 1999; 40: 1520-1528Abstract Full Text Full Text PDF PubMed Google Scholar), and microsomal oxysterol 7α-hydroxylase (CYP7B1) (29Hirayama T. Honda A. Matsuzaki Y. Miyazaki T. Ikegami T. Doy M. Xu G. Lea M. Salen G. Hypercholesterolemia in rats with hepatomas: increased oxysterols accelerate efflux but do not inhibit biosynthesis of cholesterol.Hepatology. 2006; 44: 602-611Crossref PubMed Scopus (18) Google Scholar) were measured according to our stable-isotope dilution MS method except that LC-MS/MS was used instead of GC-MS to quantify 7α-hydroxycholesterol, 27-hydroxycholesterol, 7α,27-dihydroxycholesterol, and their isotopic variants (25Honda A. Miyazaki T. Ikegami T. Iwamoto J. Yamashita K. Numazawa M. Matsuzaki Y. Highly sensitive and specific analysis of sterol profiles in biological samples by HPLC-ESI-MS/MS.J. Steroid Biochem. Mol. Biol. 2010; 121: 556-564Crossref PubMed Scopus (52) Google Scholar). Microsomal 7α-hydroxy-4-cholesten-3-one 12α-hydroxylase (CYP8B1) activity was determined as described previously (30Honda A. Salen G. Matsuzaki Y. Batta A.K. Xu G. Leitersdorf E. Tint G.S. Erickson S.K. Tanaka N. Shefer S. Side chain hydroxylations in bile acid biosynthesis catalyzed by CYP3A are markedly up-regulated in Cyp27–/– mice but not in cerebrotendinous xanthomatosis.J. Biol. Chem. 2001; 276: 34579-34585Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) except that [2H7]7α,27-dihydroxycholesterol was used as an internal standard and LC-MS/MS was employed instead of HPLC to quantify 7α,12α-dihydroxy-4-cholesten-3-one (25Honda A. Miyazaki T. Ikegami T. Iwamoto J. Yamashita K. Numazawa M. Matsuzaki Y. Highly sensitive and specific analysis of sterol profiles in biological samples by HPLC-ESI-MS/MS.J. Steroid Biochem. Mol. Biol. 2010; 121: 556-564Crossref PubMed Scopus (52) Google Scholar). An aliquot of the liver and terminal ileum specimen were collected in RNAlater (Thermo Fisher Scientific) and stored at −80°C until RNA isolation. Total RNA was extracted using an RNeasy Plus Mini Kit (QIAGEN). Reverse transcription was performed on 4 μg of total RNA using a Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany). Real-time quantitative PCR was performed on cDNA aliquots with the FastStart DNA MasterPLUS SYBR Green I and a LightCycler (Roche). The sequences of the oligonucleotide primer pairs used to amplify mRNAs are shown in supplemental Table S2. PCR amplification began with a 10 min preincubation step at 95°C, followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 62°C for 10 s, and elongation at 72°C for 16 s. The relative concentration of the PCR product derived from the target gene was calculated by the comparative Ct method, and results were standardized to the expression of Gapdh. The specificity of each PCR product was assessed by melting curve analysis. Serum concentrations of fibroblast growth factor 15 (FGF15) were measured using mouse FGF15 ELISA kit (catalog #MBS2700661; MyBiosource, Inc., San Diego, CA), according to the manufacturer's instruction. Serum concentrations of lipopolysaccharides (LPSs; endotoxin) and TNFα were quantified using ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (catalog #L00350; GenScript USA Inc., Piscataway, NJ) and LBIS Mouse TNF-α ELISA Kit (catalog #AKMTNFA-011; FUJIFILM Wako Shibayagi, Gunma, Japan), respectively, according to the manufacturers' instructions. An aliquot of the liver was fixed in 10% neutral buffered formalin and embedded in a paraffin block. Each paraffin block was sectioned at 3 μm and the paraffin sections were stained using hematoxylin/eosin. Data are expressed as the mean ± SEM. The statistical significance of differences between the results in the different groups was evaluated using the Tukey-Kramer test or the Dunnett's test. For all analyses, significance was accepted at the level of P < 0.05. Correlations were tested by calculating parametric Pearson's correlation coefficient, r, and nonparametric Spearman's correlation coefficient, rs. All statistical analyses were conducted using JMP (version 10.0) software (SAS Institute, Cary, NC). We first explored the mouse and rat genes encoding BA 7α-hydroxylase. To confirm the usefulness of our new method, genes encoding CDCA 6β-hydroxylase [Cyp2c70 is strongly suggested as a responsible gene (16Takahashi S. Fukami T. Masuo Y. Brocker C.N. Xie C. Krausz K.W. Wolf C.R. Henderson C.J. Gonzalez F.J. Cyp2c70 is responsible for the species difference in bile acid metabolism between mice and humans.J. Lipid Res. 2016; 57: 2130-2137Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar)] were also examined along with those encoding BA 7α-hydroxylase. Mouse and rat genes encoding BA 7α-hydroxylase and CDCA 6β-hydroxylase must fall under any of 102 mouse and 87 rat Cyp genes (20Nelson D.R. Gene nomenclature by default, or BLASTing to Babel.Hum. Genomics. 2005; 2: 196-201Crossref PubMed Scopus (11) Google Scholar, 21Renaud H.J. Cui J.Y. Khan M. Klaassen C.D. Tissue distribution and gender-divergent expression of 78 cytochrome P450 mRNAs in mice.Toxicol. Sci. 2011; 124: 261-277Crossref PubMed Scopus (130) Google Scholar) (Fig. 1B). Species difference, sexual dimorphism, and tissue distribution with respect to both enzymes were determined using commercially available microsomal fractions. Neither of the two enzyme activities was detected in the human liver or mouse kidney. Furthermore, sexual dimorphism of both enzyme activities was not observed in the mouse liver but was apparent in the rat liver (Fig. 1C). From the 102 mouse Cyp genes, we narrowed down to four candidate genes, Cyp2a12, Cyp2a22, Cyp2c70, and Cyp2d40 (Fig. 1D, supplemental Table S3), using previously reported orthology, tissue distribution, and sex difference data (20Nelson D.R. Gene nomenclature by default, or BLASTing to Babel.Hum. Genomics. 2005; 2: 196-201Crossref PubMed Scopus (11) Google Scholar, 21Renaud H.J. Cui J.Y. Khan M. Klaassen C.D. Tissue distribution and gender-divergent expression of 78 cytochrome P450 mRNAs in mice.Toxicol. Sci. 2011; 124: 261-277Crossref PubMed Scopus (130) Google Scholar) as well as our experimental observations (Fig. 1C). If Cyp2c70 encodes for CDCA 6β-hydroxylase (16Takahashi S. Fukami T. Masuo Y. Brocker C.N. Xie C. Krausz K.W. Wolf C.R. Henderson C.J. Gonzalez F.J. Cyp2c70 is responsible for the species difference in bile acid metabolism between mice and humans.J. Lipid Res. 2016; 57: 2130-2137Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), the gene encoding BA 7α-hydroxylase would be either Cyp2a12, Cyp2a22, or Cyp2d40. Because mouse CYP2A12, rat CYP2A1, and hamster CYP2A9 have homologous amino acid sequences and similar testosterone 7α-hydroxylase activity (31Kurose K. Tohkin M. Ushio F. Fukuhara M. Cloning and characterization of Syrian hamster testosterone 7α-hydroxylase, CYP2A9.Arch. Biochem. Biophys. 1998; 351: 60-65Crossref PubMed Scopus (14) Google Scholar), we incubated recombinant rat CYP2A1 with TDCA and TLCA. As a result, CYP2A1 catalyzed the 7α-rehydroxylation of TDCA and TLCA (Fig. 1E), suggesting that Cyp2a12 is responsible for BA 7α-rehydroxylation in mice. It is important to note that Cyp2a22 may also encode for BA 7α-hydroxylase in mice because it is highly homologous to Cyp2a12 (96.2% mRNA identity). However, the hepatic expression level of Cyp2a22 is extremely low (less than 3%) compared with that of Cyp2a12 (21Renaud H.J. Cui J.Y. Khan M. Klaassen C.D. Tissue distribution and gender-divergent expression of 78 cytochrome P450 mRNAs in mice.Toxicol. Sci. 2011; 124: 261-277Crossref PubMed Scopus (130) Google Scholar). Therefore, we finally concluded that Cyp2a12 was primarily responsible for BA 7α-rehydroxylation in mice. We generated Cyp2a12/Cyp2c70 DKO mice using the CRISPR-Cas9 system (Fig. 2A, B). Founder (F0) mice were selected by PCR genotyping (Fig. 2C, D) followed by the detection of Cas9 (supplemental Fig. S1) and direct sequencing (supplemental Table S4). Finally, #4, #5, #44, and #47 mice for Cyp2a12-null and #17, #19, and #32 mice for Cyp2c70-null were selected as F0 mice. The F0 mice were bred with WT C57BL/6J mice, and PCR genotyping followed by sequencing assay demonstrated that mice from #4, #5, and #47 lines and those from #19 line had DNA sequences at CRISPR target sites, as predicted (supplemental Table S5). Finally, a male F1 Cyp2a12+/− mouse from the #4 line and a male F1 Cyp2c70+/− mouse from the #19 line were used for subsequent