Species differences in bile acids I. Plasma and urine bile acid composition

Maintenance of bile acid (BA) homeostasis is essential to achieve their physiologic functions and avoid their toxic effects. The marked differences in BA composition between preclinical safety models and humans may play a major role in the poor prediction of drug‐induced liver injury using preclinical models. We compared the composition of plasma and urinary BAs and their metabolites between humans and several animal species. Total BA pools and their composition varied widely among different species. Highest sulfation of BAs was observed in human and chimpanzee. Glycine amidation was predominant in human, minipig, hamster and rabbit, while taurine amidation was predominant in mice, rat and dogs. BA profiles consisted primarily of tri‐OH BAs in hamster, rat, dog and mice, di‐OH BAs in human, rabbit and minipig, and mono‐OH BA in chimpanzee. BA profiles comprised primarily hydrophilic and less toxic BAs in mice, rat, pig and hamster, while it primarily comprised hydrophobic and more toxic BAs in human, rabbit and chimpanzee. Therefore, the hydrophobicity index was lowest in minipig and mice, while it was highest in rabbit, monkey and human. Glucuronidation and glutathione conjugation were low in all species across all BAs. Total concentration of BAs in urine was up to 10× higher and more hydrophilic than plasma in most species. This was due to the presence of more tri‐OH, amidated, sulfated and primary BAs, in urine compared to plasma. In general, BA profiles of chimpanzee and monkeys were most similar to human, while minipig, rat and mice were most dissimilar to human.     

Hepatotoxicity of cantharidin is associated with the altered bile acid metabolism

Cantharidin (CTD) is an effective antitumor agent. However, it exhibits significant hepatotoxicity, the mechanism of which remains unclear. In this study, biochemical and histopathological analyses complemented with ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS)-based targeted metabolomic analysis of bile acids (BAs) were employed to investigate CTD-induced hepatotoxicity in rats. Sixteen male and female Sprague–Dawley rats were randomly divided into two groups: control and CTD (1.0 mg/kg) groups. Serum and liver samples were collected after 28 days of intervention. Biochemical, histopathological, and BA metabolomic analyses were performed for all samples. Further, the key biomarkers of CTD-induced hepatotoxicity were identified via multivariate and metabolic pathway analyses. In addition, metabolite–gene–enzyme network and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to identify the signaling pathways related to CTD-induced hepatotoxicity. The results revealed significantly increased levels of biochemical indices (alanine aminotransferase, aspartate aminotransferase, and total bile acid). Histopathological analysis revealed that the hepatocytes were damaged. Further, 20 endogenous BAs were quantitated via UHPLC-MS/MS, and multivariate and metabolic pathway analyses of BAs revealed that hyocholic acid, cholic acid, and chenodeoxycholic acid were the key biomarkers of CTD-induced hepatotoxicity. Meanwhile, primary and secondary BA biosynthesis and taurine and hypotaurine metabolism were found to be associated with the mechanism by which CTD induced hepatotoxicity in rats. This study provides useful insights for research on the mechanism of CTD-induced hepatotoxicity.     

Species differences in bile acids II. Bile acid metabolism

One of the mechanisms of drug‐induced liver injury (DILI) involves alterations in bile acid (BA) homeostasis and elimination, which encompass several metabolic pathways including hydroxylation, amidation, sulfation, glucuronidation and glutathione conjugation. Species differences in BA metabolism may play a major role in the failure of currently used in vitro and in vivo models to predict reliably the DILI during the early stages of drug discovery and development. We developed an in vitro cofactor‐fortified liver S9 fraction model to compare the metabolic profiles of the four major BAs (cholic acid, chenodeoxycholic acid, lithocholic acid and ursodeoxycholic acid) between humans and several animal species. High‐ and low‐resolution liquid chromatography–tandem mass spectrometry and nuclear magnetic resonance imaging were used for the qualitative and quantitative analysis of BAs and their metabolites. Major species differences were found in the metabolism of BAs. Sulfation into 3‐O‐sulfates was a major pathway in human and chimpanzee (4.8%–52%) and it was a minor pathway in all other species (0.02%–14%). Amidation was primarily with glycine (62%–95%) in minipig and rabbit and it was primarily with taurine (43%–81%) in human, chimpanzee, dog, hamster, rat and mice. Hydroxylation was highest (13%–80%) in rat and mice followed by hamster, while it was lowest (1.6%–22%) in human, chimpanzee and minipig. C6‐β hydroxylation was predominant (65%–95%) in rat and mice, while it was at C6‐α position in minipig (36%–97%). Glucuronidation was highest in dog (10%–56%), while it was a minor pathway in all other species (<12%). The relative contribution of the various pathways involved in BA metabolism in vitro were in agreement with the observed plasma and urinary BA profiles in vivo and were able to predict and quantify the species differences in BA metabolism. In general, overall, BA metabolism in chimpanzee is most similar to human, while BA metabolism in rats and mice is most dissimilar from human.     

In vitro/in vivo investigations to examine the gender differences in the pharmacokinetics of novel oral Janus kinase (JAK) inhibitor ASP015K and sulfate metabolite M2 in rats

Abstract

1. Although marked gender differences have been reported for the exposure level of the sulfate metabolite M2 of ASP015K in rats, no such differences have been reported for the unchanged drug. To clarify the cause of these pharmacokinetic gender differences, we investigated the in vitro hepatic sulfation, glucuronidation, and cytochrome P450 (CYP) metabolism of ASP015K in rat liver cytosols or rat liver microsomes. Further, in vivo excretion and metabolic profiles were investigated using rat urine, bile, and feces post-ASP015K administration.

2. In vitro metabolism study using liver cytosols clearly suggested that the gender differences in the M2 exposure were mainly attributed to the female-predominant ASP015K metabolism mediated by sulfotransferase (SULT). Metabolic profiles in urine and bile from male rats suggested that the major elimination pathway of ASP015K is glucuronidation in rats. No remarkable gender differences in the in vitro glucuronidation were observed.

3. The contribution of the sulfation pathway to the clearance of ASP015K was markedly lower than that of the glucuronidation pathway in both male and female rats. These results might explain why gender differences were not marked for ASP015K exposure but were for M2.     

液-质联用测定Nrf2野生型和基因敲除型小鼠肝脏中16种胆汁酸

目的:建立测定小鼠肝脏中16种胆汁酸浓度的LC-MS/MS法,并将测定结果应用于比较Nrf2野生型和基因敲除型小鼠胆汁酸代谢谱。方法:小鼠肝脏样品加入70%乙腈匀浆,用甲醇沉淀匀浆液中的蛋白并高速离心,取上清液置进样瓶, 用Xtimate^TM C₁₈柱分离,以甲醇-醋酸铵及甲酸水溶液为流动相进行梯度洗脱,流速0.25 ml·min^-1,进样5 μl分析。结果:16种胆汁酸在0.013~8.000 μmol·L^-1范围内,线性关系良好,定量下限浓度为0.013 μmol·L^-1,肝脏匀浆液加样回收率在94.9%~112.8%之间,日内、日间RSD均小于10.8%。结论:该方法准确可靠,灵敏度高,处理简便,适用于肝脏中胆汁酸浓度的测定,为生物样品中胆汁酸含量测定提供参考。     

采用UFLC-IT-TOF/MS鉴定大鼠口服黄芩提取物后胆汁、血浆和尿液中的主要成分(英文)

黄酮类化合物是中国传统中药黄芩的主要活性成分。大鼠口服黄芩提取物后, 采用超快速液相色谱-离子阱飞行时间质谱(UFLC-IT-TOF/MS)对胆汁、血浆和尿液进行分析, 鉴定入血成分和代谢产物。根据对这些成分质谱裂解规律的分析, 共鉴定了36种不同的黄酮类化合物, 其中包括13个新的代谢产物。本实验在体外对黄芩提取物进行分析,鉴定了其中16个黄酮类成分, 大鼠胆汁中鉴定了25个黄酮类化合物, 血浆中15个黄酮类化合物, 尿液中14个黄酮类化合物。结果表明, 黄芩提取物中的黄酮类化合物以葡萄糖醛酸化、硫酸化和甲基化的为主要代谢途径。本实验首次对黄芩提取物的代谢产物进行综合分析。     

Acetaminophen-induced hepatotoxicity in rats is correlated with the accumulation of bile acids: an underlying mechanism

In the present study, we aimed to investigate the underlying mechanism of acetaminophen (APAP)-induced hepatotoxicityby measuring the expression levels of liver transporters and concentrations of bile acids (BAs) in rat plasma and liver. SD rats (42)were randomly assigned into six groups, including 6-h control group, APAP 6-h group, 12-h control group, APAP 12-h group, 24-h control group and APAP 24-h group. The estimation study of BAs in plasma and liver was performed on LC-MS/MS.The levels of bile salt export pump (Bsep), multidrug resistant protein 2 (Mrp2), multidrug resistant protein 4 (Mrp4), Na⁺/taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide 2 (Oatp2) in the liver were analyzed by Western blotting analysis. Compared with the corresponding control groups, no difference was found in the BA levels and the expressions of BA transporters in the plasma and liver after 6 h of APAP administration. While BA levels were significantly decreased in the plasma and increased in the liver after 12 h of APAP administration (P<0.05); and the expressions of Bsep and Mrp2 were significantly reduced (P<0.05). After 24 h of APAP administration, BA levels were both greatly increased in the plasma and liver (P<0.05); and the expressions of Mrp4 and Oatp2 were significantly decreased (P<0.05). In response to over-dose APAP, Bsep, Mrp2, Mrp4 and Oatp2 levels were reduced at different time points, causing the accumulation of BAs, and such accumulation may ultimately lead to the severe liver injury, which could be an underlying mechanism of the APAP-induced hepatotoxicity.     

Increased bile acids in enterohepatic circulation by short-term calorie restriction in male mice

Previous studies showed glucose and insulin signaling can regulate bile acid (BA) metabolism during fasting or feeding. However, limited knowledge is available on the effect of calorie restriction (CR), a well-known anti-aging intervention, on BA homeostasis. To address this, the present study utilized a “dose–response” model of CR, where male C57BL/6 mice were fed 0, 15, 30, or 40% CR diets for one month, followed by BA profiling in various compartments of the enterohepatic circulation by UPLC-MS/MS technique. This study showed that 40% CR increased the BA pool size (162%) as well as total BAs in serum, gallbladder, and small intestinal contents. In addition, CR “dose-dependently” increased the concentrations of tauro-cholic acid (TCA) and many secondary BAs (produced by intestinal bacteria) in serum, such as tauro-deoxycholic acid (TDCA), DCA, lithocholic acid, ω-muricholic acid (ωMCA), and hyodeoxycholic acid. Notably, 40% CR increased TDCA by over 1000% (serum, liver, and gallbladder). Interestingly, 40% CR increased the proportion of 12α-hydroxylated BAs (CA and DCA), which correlated with improved glucose tolerance and lipid parameters. The CR-induced increase in BAs correlated with increased expression of BA-synthetic (Cyp7a1) and conjugating enzymes (BAL), and the ileal BA-binding protein (Ibabp). These results suggest that CR increases BAs in male mice possibly through orchestrated increases in BA synthesis and conjugation in liver as well as intracellular transport in ileum.     

LC-MS/MS法同时测定ICR小鼠肝脏中15种胆汁酸及其在雷公藤甲素肝毒性研究中的应用

目的：建立同时测定ICR小鼠肝脏中15种胆汁酸的LC-MS/MS法,将其应用到雷公藤甲素肝毒性研究中。方法：小鼠经腹腔注射1 mg·kg^-1雷公藤甲素后,进行血清生化、肝病理切片检查。将肝脏用0.1%甲酸-75%乙腈混合液进行匀浆并经样品处理,采用Ultimate^® AQ-C₁₈色谱柱,流速0.3 mL·min^-1,进行15种胆汁酸色谱分离。结果：LC-MS/MS法测定的15种胆汁酸在2.8~8 000.0 nmol·L^-1浓度范围内具有较好的线性关系,加样回收率在85.8%~114.0%之间,日内、日间精密度均在0.7%~10.8%范围内;腹腔注射1 mg·kg^-1雷公藤甲素后小鼠出现了肝毒性,雷公藤甲素组的胆汁酸浓度有明显上升。结论：该方法准确、稳定性好、灵敏度高,能满足ICR小鼠肝脏中多种胆汁酸的含量测定,为雷公藤甲素肝毒性研究提供了进一步的理论基础。     

Metabolite profiling of guanfacine in plasma and urine of healthy Japanese subjects after oral administration of guanfacine extended‐release tablets

Guanfacine is used for the treatment of attention‐deficit/hyperactivity disorder (ADHD). Using liquid chromatography–tandem mass spectrometry (LC–MS/MS), metabolite profiling of guanfacine was performed in plasma and urine collected from healthy Japanese adults following repeated oral administration of guanfacine extended‐release formulation. Unchanged guanfacine was the most abundant component in both plasma and urine (from the MS signal intensity). In plasma, the M3 metabolite (a sulfate of hydroxy‐guanfacine) was the prominent metabolite; the M2 metabolite (a glucuronide of a metabolite formed by monooxidation of guanfacine), 3‐hydroxyguanfacine and several types of glucuronide at different positions on guanfacine were also detected. In urine, the M2 metabolite and 3‐hydroxyguanfacine were the principal metabolites. From metabolite analysis, the proposed main metabolic pathway of guanfacine is monooxidation on the dichlorobenzyl moiety, followed by glucuronidation or sulfation. A minor pathway is glucuronidation at different positions on guanfacine. As the prominent metabolites in plasma were glucuronide and sulfate of hydroxyguanfacine, which have no associated toxicity concerns, further toxicity studies of the metabolites, for example in animals, were not deemed necessary.     

采用UHPLC-MS和质量亏损过滤技术分析二苯乙烯苷在大鼠体内的代谢产物和代谢途径

目的:分析二苯乙烯苷在大鼠体内的代谢产物并推测代谢途径。方法:将雄性SD大鼠随机分为血浆组(n=3)、尿液组(n=3)、胆汁组(n=3)和组织组(n=9),各组大鼠均单次灌胃二苯乙烯苷200 mg/kg,分别收集给药后10、30 min和1、1.5、2、4 h的血浆,给药后0~6 h的尿液,给药后0~4 h的胆汁以及给药后30 min和1、2 h(每个时间点3只)的心、肝、脾、肺、肾、胃组织样品,经甲醇沉淀蛋白后,采用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱联用技术和质量亏损过滤技术联合分析、鉴定各样本中的代谢产物,推测代谢途径。结果:从血浆、尿液、胆汁、心、肝、脾、肺、肾、胃样品中分别检出6、7、11、1、5、1、3、4、4个代谢产物,包括Ⅰ相代谢(如水解、加氢、羟化)产物2个、Ⅱ相代谢(如葡萄糖醛酸结合和硫酸化)产物18个,其中葡萄糖醛酸结合产物有12个。结论:二苯乙烯苷在胆汁中的代谢产物种类居多,以Ⅱ相代谢产物二苯乙烯苷的葡萄糖醛酸结合产物为主;代谢途径主要涉及葡萄糖水解、加氢、羟化、葡萄糖醛酸结合、硫酸化反应等。     

应用HPLC-MS技术测定健康受试者和肝癌患者血浆中13种胆汁酸的含量

目的建立HPLC-MS方法同时检测健康人和原发性肝癌患者血浆中13种胆汁酸的含量。方法血浆样品前处理采用乙腈-蛋白沉淀法,色谱分离采用SHISEIDO MG C18（100 mm×3.0 mm,3.0μm）柱,流动相：乙腈-水（含0.1%甲酸和0.5%氨水）,梯度洗脱,流速0.4 ml/min,柱温25℃,进样量5μl。质谱监测采用ESI源负离子模式,选择离子（SIM）分段监测。结果 13种胆汁酸在3.3~4 400 ng/ml的浓度范围内线性关系良好;日内和日间精密度均〈7%;平均回收率为82.67%~109.75%。样品测定结果显示,与健康对照组相比,原发性肝癌患者血浆中胆汁酸含量升高,其中以结合型胆汁酸TUDCA、TCDCA、TCA、GCA和GCDCA的升高最为明显。结论所建HPLC-MS方法灵敏度高、选择性好,可用于原发性肝癌患者与健康人体内血清胆汁酸的含量差异比较,该研究为肝癌患者体内胆汁酸的代谢和疾病的临床诊治奠定了基础。     

Novel insights into bile acid detoxification via CYP,UGT and SULT enzymes

Bile acid (BA) homeostasis is a complex and precisely regulated process to prevent impaired BA flow and the development of cholestasis. Several reactions, namely hydroxylation, glucuronidation and sulfation are involved in BA detoxification. In the present study, we employed a comprehensive approach to identify the key enzymes involved in BA metabolism using human recombinant enzymes, human liver microsomes (HLM) and human liver cytosol (HLC). We showed that CYP3A4 was a crucial step for the metabolism of several BAs and their taurine and glycine conjugated forms and quantitatively described their metabolites. Glucuronidation and sulfation were also identified as important drivers of the BA detoxification process in humans. Moreover, lithocholic acid (LCA), the most hydrophobic BA with the highest toxicity potential, was a substrate for all investigated processes, demonstrating the importance of hepatic metabolism for its clearance. Collectively, this study identified CYP3A4, UGT1A3, UGT2B7 and SULT2A1 as the major contributing (metabolic) processes in the BA detoxification network. Inhibition of these enzymes by drug candidates is therefore considered as a critical mechanism in the manifestation of drug-induced cholestasis in humans and should be addressed during the pre-clinical development.     

基于胆汁酸代谢谱对自身免疫性肝炎小鼠模型的早期预判研究

目的:采用液相色谱质谱联用仪(LC-MS/MS)测定胆汁酸代谢谱的分析方法评价刀豆蛋白A诱导自身免疫性肝炎(autoimmune hepatitis,AIH)小鼠模型.方法:小鼠经30 mg· kg-1刀豆蛋白A诱导后可成功建立AIH模型(可称为模型组);而经20 mg· kg-1刀豆蛋白A诱导后其生化、病理指标均未发...     

A novel constitutive androstane receptor-mediated and CYP3A-independent pathway of bile acid detoxification

Cytosolic sulfotransferase (SULT)-mediated sulfation plays an essential role in the detoxification of bile acids and is necessary to avoid pathological conditions, such as cholestasis, liver damage, and colon cancer. In this study, using transgenic mice bearing conditional expression of the activated constitutive androstane receptor (CAR), we demonstrate that activation of CAR is both necessary and sufficient to confer resistance to the hepatotoxicity of lithocholic acid (LCA). Surprisingly, the CAR-mediated protection is not attributable to the expected and previously characterized CYP3A pathway; rather, it is associated with a robust induction of SULT gene expression and increased LCA sulfation. We have also provided direct evidence that CAR regulates SULT expression by binding to the CAR response elements found within the SULT gene promoters. Interestingly, activation of CAR was also associated with an increased expression of the 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2), an enzyme responsible for generating the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate. Analysis of gene knockout mice revealed that CAR is also indispensable for ligand-dependent activation of SULT and PAPSS2 in vivo. Therefore, we establish an essential and unique role of CAR in controlling the mammalian sulfation system and its implication in the detoxification of bile acids.     

Molecular determinants of gastrointestinal and liver cancers: role of bile acid activated nuclear receptors

Invasion and metastasis are critical determinants of gastrointestinal and liver cancers morbidity. Genes and molecules participating in these steps (e.g. growth factors and their receptors, cell cycle regulators, cell adhesion molecules, matrix degrading enzymes) have been progressively clarified. Activated Wnt signaling pathway has been found in these tumors. Mutations in one of the DNA mismatch repair genes, alterations in epigenetics, such as aberrant DNA methylation and histone modifications are associated with the progression of gastrointestinal and control liver neoplasies. Bile acids (BAs), the main constituents of bile, activate a family of nuclear receptors (NRs) that control critical steps in bile acid homeostasis, endo- and xenobiotics detoxification, glucose, lipid metabolism and innate immunity. BAs activated NRs are misregulated in gastrointestinal and liver cancers. The present review provides an overview on the molecular determinants involved in gastrointestinal and liver cancers and focuses on the role of BAs activated NRs in the pathogenesis of these tumors.     

Simultaneous quantification of glycine- and taurine-conjugated bile acids,total bile acids,and choline-containing phospholipids in human bile using H NMR spectroscopy

Bile acids, phospholipids, and cholesterol are the major lipid components in human bile. The composition of bile is altered in various cholestatic diseases, and determining such alterations will be of great clinical importance in understanding the pathophysiology of these diseases. A robust method for the simultaneous quantification of major biliary lipids – glycine-conjugated bile acids (GCBAs), taurine-conjugated bile acids (TCBAs), total bile acids (TBAs) and choline-containing phospholipids (choline-PLs) has been devised using ¹H NMR spectroscopy. Bile samples were obtained from patients with various hepatopancreatobiliary diseases (n = 10) during an endoscopic retrograde cholangiopancreatography (ERCP) examination. Peak areas of metabolite-signals of interest were obtained simultaneously by deconvoluting the experimental spectrum, making the present method robust. GCBAs and TCBAs have been quantified using the peak areas of their characteristic methylene (CH₂) signals resonating at 3.73 and 3.07 ppm, whereas TBA and choline-PLs were quantified using their methyl (CH₃) and trimethylammonium (–N⁺(CH₃)₃) signals resonating at 0.65 and 3.22 ppm respectively. The present method was compared with an NMR-based literature method (which involves dissolving bile in DMSO), and a good correlation was observed between the two methods with regression coefficients – 0.97, 0.99, 0.98 and 0.93 for GCBAs, TCBAs, TBAs, and choline-PLs respectively. This method has the potential to be extended to in vivo applications for the simultaneous quantification of various biliary lipids non-invasively.     

HPLC assay of conjugated bile acids in gastric juice during ursodeoxycholic acid (Deursil) therapy of bile reflux gastritis

A rapid high-performance liquid chromatographic method for the direct assay of the taurine and glycine conjugated bile acids in human gastric juice is described. After extraction with Sep-Pak C₁₈ cartridges, compounds are baseline resolved on a reversed-phase column and detected by UV absorption. The procedure is linear from 10 μmol l⁻¹ to 1200 μmol l⁻¹, with recovery rates ranging from 87 to 100%. The present method is applicable to the quantification of bile acid conjugates in human gastric bile with satisfactory sensitivity, selectivity and precision. Intragastric bile acid compositions in 10 patients with bile reflux gastritis during Deursil® or placebo treatment are presented.     

Regulation of hepatic bile acid transporters Ntcp and Bsep expression

Sodium-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) are two key transporters for hepatic bile acid uptake and excretion. Alterations in Ntcp and Bsep expression have been reported in pathophysiological conditions. In the present study, the effects of age, gender, and various chemicals on the regulation of these two transporters were characterized in mice. Ntcp and Bsep mRNA levels in mouse liver were low in the fetus, but increased to its highest expression at parturition. After birth, mouse Ntcp and Bsep mRNA decreased by more than 50%, and then gradually increased to adult levels by day 30. Expression of mouse Ntcp mRNA and protein exhibit higher levels in female than male livers. No gender difference exists in BSEP/Bsep expression in human and mouse livers. Hormone replacements conducted in gonadectomized, hypophysectomized, and lit/lit mice indicate that female-predominant Ntcp expression in mouse liver is due to the inhibitory effect of male-pattern GH secretion, but not sex hormones. Ntcp and Bsep expression are in general resistant to induction by a large battery of microsomal enzyme inducers. Administration of cholestyramine increased Ntcp, whereas chenodeoxycholic acid (CDCA) increased Bsep mRNA expression. In conclusion, mouse Ntcp and Bsep are regulated by age, gender, cholestyramine, and bile acid, but resistant to induction by most microsomal enzyme inducers.     

Effect of various antibiotics on modulation of intestinal microbiota and bile acid profile in mice

Antibiotic treatments have been used to modulate intestinal bacteria and investigate the role of intestinal bacteria on bile acid (BA) homeostasis. However, knowledge on which intestinal bacteria and bile acids are modified by antibiotics is limited. In the present study, mice were administered various antibiotics, 47 of the most abundant bacterial species in intestine, as well as individual BAs in plasma, liver, and intestine were quantified. Compared to the two antibiotic combinations (vancomycin + imipenem and cephalothin + neomycin), the three single antibiotics (metronidazole, ciprofloxacin and aztreonam) have less effect on intestinal bacterial profiles, and thus on host BA profiles and mRNA expression of genes that are important for BA homeostasis. The two antibiotic combinations decreased the ratio of Firmicutes to Bacteroidetes in intestine, as well as most secondary BAs in serum, liver and intestine. Additionally, the two antibiotic combinations significantly increased mRNA of the hepatic BA uptake transporters (Ntcp and Oatp1b2) and canalicular BA efflux transporters (Bsep and Mrp2), but decreased mRNA of the hepatic BA synthetic enzyme Cyp8b1, suggesting an elevated enterohepatic circulation of BAs. Interestingly, the two antibiotic combinations tended to have opposite effect on the mRNAs of most intestinal genes, which tended to be inhibited by vancomycin + imipenem but stimulated by cephalothin + neomycin. To conclude, the present study clearly shows that various antibiotics have distinct effects on modulating intestinal bacteria and host BA metabolism.