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.     

Alpha-naphthylisothiocyanate modulates hepatobiliary transporters in sandwich-cultured rat hepatocytes

Alpha-naphthylisothiocyanate (ANIT) induces intra-hepatic cholestasis mixed with hepatocellular injury mainly by bile ductular damage. However, its direct effect on hepatic parenchymal cells (hepatocytes) is unclear. Sandwich-cultured rat hepatocytes (SCRH) were applied to clarify this question. Though cytotoxicity was not observed (0–180 μM) in ANIT-treated SCRH, metabonomics analysis of the hepatocytes revealed a shift in the metabolic pattern and a decrease in cellular cholesterol level, accompanied by an increase in total bile acids after 48 h ANIT (5–45 μM) treatment. To assess the function of major hepatic bile acid transporters, the accumulation and efflux of [D-Pen^2,5]-enkephalin (DPDPE), 5 (and 6)-carboxy-2′,7′-dichlorofluorescein (CDF) diacetate promoiety and deuterium-labeled sodium taurocholate (d8-TCA) were measured. ANIT incubation for either 30 min or 48 h led to dose-dependent decreases in the biliary excretion index (BEI) of DPDPE and CDF, as well as the intracellular accumulation of d8-TCA, CDF and DPDPE. The basolateral efflux of d8-TCA was also decreased with its BEI barely changed. mRNA expression of multiple uptake transporters and bile acid synthesizing enzymes was down-regulated after 48 h incubation. In conclusion, ANIT could directly induce retention of bile acids in hepatocytes by inhibiting the function of bile acid transporters, which might contribute to its cholestatic effect.     

Oleanolic acid alters bile acid metabolism and produces cholestatic liver injury in mice

Oleanolic acid (OA) is a triterpenoids that exists widely in plants. OA is effective in protecting against hepatotoxicants. Whereas a low dose of OA is hepatoprotective, higher doses and longer-term use of OA produce liver injury. This study characterized OA-induced liver injury in mice. Adult C57BL/6 mice were given OA at doses of 0, 22.5, 45, 90, and 135 mg/kg, s.c., daily for 5 days, and liver injury was observed at doses of 90 mg/kg and above, as evidenced by increases in serum activities of alanine aminotransferase and alkaline phosphatase, increases in serum total bilirubin, as well as by liver histopathology. OA-induced cholestatic liver injury was further evidenced by marked increases of both unconjugated and conjugated bile acids (BAs) in serum. Gene and protein expression analysis suggested that livers of OA-treated mice had adaptive responses to prevent BA accumulation by suppressing BA biosynthetic enzyme genes (Cyp7a1, 8b1, 27a1, and 7b1); lowering BA uptake transporters (Ntcp and Oatp1b2); and increasing a BA efflux transporter (Ostβ). OA increased the expression of Nrf2 and its target gene, Nqo1, but decreased the expression of AhR, CAR and PPARα along with their target genes, Cyp1a2, Cyp2b10 and Cyp4a10. OA had minimal effects on PXR and Cyp3a11. Taken together, the present study characterized OA-induced liver injury, which is associated with altered BA homeostasis, and alerts its toxicity potential.     

Hepatocyte-based in vitro model for assessment of drug-induced cholestasis

Early detection of drug-induced cholestasis remains a challenge during drug development. We have developed and validated a biorelevant sandwich-cultured hepatocytes- (SCH) based model that can identify compounds causing cholestasis by altering bile acid disposition. Human and rat SCH were exposed (24–48 h) to known cholestatic and/or hepatotoxic compounds, in the presence or in the absence of a concentrated mixture of bile acids (BAs). Urea assay was used to assess (compromised) hepatocyte functionality at the end of the incubations. The cholestatic potential of the compounds was expressed by calculating a drug-induced cholestasis index (DICI), reflecting the relative residual urea formation by hepatocytes co-incubated with BAs and test compound as compared to hepatocytes treated with test compound alone. Compounds with clinical reports of cholestasis, including cyclosporin A, troglitazone, chlorpromazine, bosentan, ticlopidine, ritonavir, and midecamycin showed enhanced toxicity in the presence of BAs (DICI ≤ 0.8) for at least one of the tested concentrations. In contrast, the in vitro toxicity of compounds causing hepatotoxicity by other mechanisms (including diclofenac, valproic acid, amiodarone and acetaminophen), remained unchanged in the presence of BAs. A safety margin (SM) for drug-induced cholestasis was calculated as the ratio of lowest in vitro concentration for which was DICI ≤ 0.8, to the reported mean peak therapeutic plasma concentration. SM values obtained in human SCH correlated well with reported % incidence of clinical drug-induced cholestasis, while no correlation was observed in rat SCH. This in vitro model enables early identification of drug candidates causing cholestasis by disturbed BA handling.     

Intestinal drug transporters: An overview

The importance of drug transporters as one of the determinants of pharmacokinetics has become increasingly evident [1]. While much research has been conducted focusing the role of drug transporters in the liver , ,  and  and kidney ,  and  less is known about the importance of uptake and efflux transporters identified in the intestine [8]. Over the past years the effects of intestinal transporters have been studied using in vivo models, in situ organ perfusions, in vitro tissue preparations and cell lines. This review aims to describe up to date findings regarding the importance of intestinal transporters on drug absorption and bioavailability, highlighting areas in need of further research. Wu and Benet [9] proposed a Biopharmaceutics Drug Disposition Classification System (BDDCS) that allows the prediction of transporter effects on the drug disposition of orally administered drugs. This review also discusses BDDCS predictions with respect to the role of intestinal transporters and intestinal transporter-metabolizing enzyme interplay on oral drug pharmacokinetics.     

Drug metabolizing enzyme and transporter protein profiles of hepatocytes derived from human embryonic and induced pluripotent stem cells

Human embryonic and induced pluripotent stem cell-derived hepatocytes (hESC-Hep and hiPSC-Hep) have the potential to provide relevant human in vitro model systems for toxicity testing and drug discovery studies. In this study, the expression and function of important drug metabolizing cytochrome P450 (CYP) enzymes and transporter proteins in hESC-Hep and hiPSC-Hep were compared to cryopreserved human primary hepatocytes (hphep) and HepG2 cells. Overall, CYP activities in hESC-Hep and hiPSC-Hep were much lower than in hphep cultured for 4 h, but CYP1A and 3A activities were comparable to levels in hphep cultured for 48 h (CYP1A: 35% and 26% of 48 h hphep, respectively; CYP3A: 80% and 440% of 48 h hphep, respectively). Importantly, in hESC-Hep and hiPSC-Hep, CYP activities were stable or increasing for at least one week in culture which was in contrast to the rapid loss of CYP activities in cultured hphep between 4 and 48 h after plating. With regard to transporters, in hESC-Hep and hiPSC-Hep, pronounced NTCP activity (17% and 29% of 4 h hphep, respectively) and moderate BSEP activity (6% and 8% of 4 h hphep, respectively) were observed. Analyses of mRNA expression and immunocytochemistry supported the observed CYP and transporter activities and showed expression of additional CYPs and transporters. In conclusion, the stable expression and function of CYPs and transporters in hESC-Hep and hiPSC-Hep for at least one week opens up the possibility to reproducibly perform long term and extensive studies, e.g. chronic toxicity testing, in a stem cell-derived hepatic system.     

Betulinic Acid Ameliorates the T-2 Toxin-Triggered Intestinal Impairment in Mice by Inhibiting Inflammation and Mucosal Barrier Dysfunction through the NF-κB Signaling Pathway

T-2 toxin, a trichothecene mycotoxin produced by Fusarium, is widely distributed in crops and animal feed and frequently induces intestinal damage. Betulinic acid (BA), a plant-derived pentacyclic lupane-type triterpene, possesses potential immunomodulatory, antioxidant and anti-inflammatory biological properties. The current study aimed to explore the protective effect and molecular mechanisms of BA on intestinal mucosal impairment provoked by acute exposure to T-2 toxin. Mice were intragastrically administered BA (0.25, 0.5, or 1 mg/kg) daily for 2 weeks and then injected intraperitoneally with T-2 toxin (4 mg/kg) once to induce an intestinal impairment. BA pretreatment inhibited the loss of antioxidant capacity in the intestine of T-2 toxin-treated mice by elevating the levels of CAT, GSH-PX and GSH and reducing the accumulation of MDA. In addition, BA pretreatment alleviated the T-2 toxin-triggered intestinal immune barrier dysregulation by increasing the SIgA level in the intestine at dosages of 0.5 and 1 mg/kg, increasing IgG and IgM levels in serum at dosages of 0.5 and 1 mg/kg and restoring the intestinal C3 and C4 levels at a dosage of 1 mg/kg. BA administration at a dosage of 1 mg/kg also improved the intestinal chemical barrier by decreasing the serum level of DAO. Moreover, BA pretreatment improved the intestinal physical barrier via boosting the expression of ZO-1 and Occludin mRNAs and restoring the morphology of intestinal villi that was altered by T-2 toxin. Furthermore, treatment with 1 mg/kg BA downregulated the expression of p-NF-κB and p-IκB-α proteins in the intestine, while all doses of BA suppressed the pro-inflammatory cytokines expression of IL-1β, IL-6 and TNF-α mRNAs and increased the anti-inflammatory cytokine expression of IL-10 mRNA in the intestine of T-2 toxin-exposed mice. BA was proposed to exert a protective effect on intestinal mucosal disruption in T-2 toxin-stimulated mice by enhancing the intestinal antioxidant capacity, inhibiting the secretion of inflammatory cytokines and repairing intestinal mucosal barrier functions, which may be associated with BA-mediated inhibition of the NF-κB signaling pathway activation.     

Impact of OATP transporters on pharmacokinetics

Membrane transporters are now recognized as important determinants of the transmembrane passage of drugs. Organic anion transporting polypeptides (OATP) form a family of influx transporters expressed in various tissues important for pharmacokinetics. Of the 11 human OATP transporters, OATP1B1, OATP1B3 and OATP2B1 are expressed on the sinusoidal membrane of hepatocytes and can facilitate the liver uptake of their substrate drugs. OATP1A2 is expressed on the luminal membrane of small intestinal enterocytes and at the blood-brain barrier, potentially mediating drug transport at these sites. Several clinically used drugs have been identified as substrates of OATP transporters (e.g. many statins are substrates of OATP1B1). Some drugs may inhibit OATP transporters (e.g. cyclosporine) causing pharmacokinetic drug–drug interactions. Moreover, genetic variability in genes encoding OATP transporters can result in marked inter-individual differences in pharmacokinetics. For example, a single nucleotide polymorphism (c.521T > C, p.Val174Ala) in the SLCO1B1 gene encoding OATP1B1 decreases the ability of OATP1B1 to transport active simvastatin acid from portal circulation into the liver, resulting in markedly increased plasma concentrations of simvastatin acid and an enhanced risk of simvastatin-induced myopathy. SLCO1B1 polymorphism also affects the pharmacokinetics of many other, but not all (fluvastatin), statins and that of the antidiabetic drug repaglinide, the antihistamine fexofenadine and the endothelin A receptor antagonist atrasentan. This review compiles the current knowledge about the expression and function of human OATP transporters, their substrate and inhibitor specificities, as well as pharmacogenetics.     

Caffeine-induced inhibition of the activity of glutamate transporter type 3 expressed in Xenopus oocytes

Caffeine has been known to trigger seizures, however, the precise mechanism about the proconvulsive effect of caffeine remains unclear. Glutamate transporters play an important role to maintain the homeostasis of glutamate concentration in the brain tissue. Especially, dysfunction of excitatory amino acid transporter type 3 (EAAT3) can lead to seizures. We investigated the effects of caffeine on the activity of EAAT3 and the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K). Rat EAAT3 was expressed in Xenopus oocytes by injecting EAAT3 mRNA. l-Glutamate (30 μM)-induced inward currents were recorded via the two-electrode voltage clamp method. Caffeine decreased EAAT3 activity in a dose-dependent manner. Caffeine (30 μM for 3 min) significantly reduced V_max, but did not alter K_m value of EAAT3 for glutamate. When preincubated oocytes with phorbol-12-myristate-13-acetate (PMA, a PKC activator) were exposed to caffeine, PMA-induced increase in EAAT3 activity was abolished. Two PKC inhibitors (chelerythrine and staurosporine) significantly reduced basal EAAT3 activity. Whereas, there were no significant differences among the PKC inhibitors, caffeine, and PKC inhibitors + caffeine groups. In similarly fashion, wortmannin (a PI3K inhibitor) significantly decreased EAAT3 activity, however no statistical differences were observed among the wortmannin, caffeine, and wortmannin + caffeine groups. Our results demonstrate that caffeine attenuates EAAT3 activity and this reducing effect of caffeine seems to be mediated by PKC and PI3K.     

Timosaponin A3 induces hepatotoxicity in rats through inducing oxidative stress and down-regulating bile acid transporters

Aim:

To investigate the mechanisms underlying the hepatotoxicity of timosaponin A3 (TA3), a steroidal saponin from Anemarrhena asphodeloides, in rats.

Methods:

Male SD rats were administered TA3 (100 mg·kg⁻¹·d⁻¹, po) for 14 d, and the blood and bile samples were collected after the final administration. The viability of a sandwich configuration of cultured rat hepatocytes (SCRHs) was assessed using WST-1. Accumulation and biliary excretion index (BEI) of d8-TCA in SCRHs were determined with LC-MS/MS. RT-PCR and Western blot were used to analyze the expression of relevant genes and proteins. ROS and ATP levels, and mitochondrial membrane potential (MMP) were measured. F-actin cytoskeletal integrity was assessed under confocal microscopy.

Results:

TA3 administration in rats significantly elevated the total bile acid in serum, and decreased bile acid (BA) component concentrations in bile. TA3 inhibited the viability of the SCRHs with an IC₅₀ value of 15.21±1.73 μmol/L. Treatment of the SCRHs with TA3 (1–10 μmol/L) for 2 and 24 h dose-dependently decreased the accumulation and BEI of d8-TCA. The TA3 treatment dose-dependently decreased the expression of BA transporters Ntcp, Bsep and Mrp2, and BA biosynthesis related Cyp7a1 in hepatocytes. Furthermore, the TA3 treatment dose-dependently increased ROS generation and HO-1 expression, decreased the ATP level and MMP, and disrupted F-actin in the SCRHs. NAC (5 mmol/L) significantly ameliorated TA3-induced effects in the SCRHs, whereas mangiferin (10–200 μg/mL) almost blocked TA3-induced ROS generation.

Conclusion:

TA3 triggers liver injury through inducing ROS generation and suppressing the expression of BA transporters. Mangiferin, an active component in Anemarrhena, may protect hepatocytes from TA3-induced hepatotoxicity.     

Dynamic pattern of gene expression of ZnT-1, ZnT-3 and PRG-1 in rat brain following flurothyl-induced recurrent neonatal seizures

Zinc transporters (ZnTs) and plasticity-related genes (PRGs) both play the key roles in the formation of hippocampal mossy fiber sprouting, which is associated with cognitive deficits following developmental seizures. Here, for the first time, we report the timing of expression pattern of ZnT-1, ZnT-3 and PRG-1 in hippocampus and cerebral cortex following developmental seizures. A seizure was induced by inhalant flurothyl daily in neonatal Sprague–Dawley rats from postnatal day 6 (P6). Rats were assigned into the recurrent-seizure group (RS, seizures induced in 6 consecutive days) and the control group. At 1.5 h, 3 h, 6 h, 12 h, 24 h, 48 h, 7 d and 14 d after the last seizures, the mRNA level was detected using RT-PCR method; PRG-1 protein level was examined by Western blotting analysis. At an early period of 12 h and 48 h after the last seizures, both ZnT-1 and ZnT-3 showed significantly down-regulated mRNA level in the cerebral cortex of RS group than those at the corresponding time point in control group. In the long-term time point of 14 d after the last seizure, ZnT-3 mRNA and PRG-1 protein level in hippocampus were up-regulated while the mRNA level of ZnT-1 down-regulated; in addition, there were up-regulated level of both the mRNA and protein level of PRG-1 and down-regulated mRNA level of ZnT-3 in the cerebral cortex of RS group when compared to the control. Taken together, these dates are consistent with an important role for ZnT-1, ZnT-3 and PRG-1 in the pathophysiology of the long-term adverse effects of recurrent neonatal seizure-induced hippocampal mossy fiber sprouting and cognitive deficit.     

Influx and efflux transport as determinants of melphalan cytotoxicity: Resistance to melphalan in MDR1 overexpressing tumor cell lines

There is a considerable variation in efficacy of melphalan therapy in multiple myeloma (MM) and other hematopoietic tumors. We hypothesized that this may be due to variations in the expression of influx and efflux transporters of melphalan. We measured the expression of the influx transporters LAT1, LAT2, and TAT1 and the efflux transporters MDR1, MRP1 and BCRP by quantitative RT-PCR and related their expression to the intracellular accumulation and cytotoxicity of melphalan in 7 MM and 21 non-MM hematopoietic tumor cell lines. Variation in the intracellular accumulation accounted for nearly half of the variation in the cytotoxicity of melphalan in MM cell lines (r² = 0.47, P = 0.04). High expression of the efflux transporter MDR1 was associated with low intracellular accumulation and low cytotoxicity of melphalan (r² = 0.56, P = 0.03 and r² = 0.62, P = 0.02, respectively). The effect was reversed by the MDR1 inhibitor cyclosporine. In addition, the MDR1 overexpressing HL-60 cell line showed 10-fold higher resistance to melphalan than the non-MDR1 expressing one. Again, the resistance was reversed by cyclosporine and by MDR1-specific shRNA.LAT1 was the major influx transporter in tumor cell lines with 4000-fold higher expression than LAT2. Down-regulation of LAT1 by siRNA reduced the melphalan uptake by 58% and toxicity by 3.5-fold, but natural variation in expression between the tumor cell lines was not associated with accumulation or cytotoxicity of melphalan. In conclusion, tumor-specific variations in the expression of the efflux transporter MDR1, but not of the influx transporter LAT1, affect the intracellular accumulation of melphalan and thus determine its cytotoxicity.     

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.     

Determination of major boswellic acids in plasma by high-pressure liquid chromatography/mass spectrometry

Until now, dexamethasone is the medication of choice to reduce peritumoral edema associated with primary and secondary brain tumors. Because of the severe side effects accompanying such a treatment the interest in alternative agents that may be co-administered with glucocorticoids and help to reduce the required dose is constantly increasing. Boswellia serrata gum resin extracts (BSE), which have been designated an orphan drug status by the European Medicines Agency (EMA) in 2002 for the treatment of peritumoral edema, may represent a promising supplemental herbal remedy. However, clinical studies on the effect of BSE on brain edema as well as analyzes of serum levels are very scarce. Based on that background a prospective, placebo controlled, and double blind clinical pilot trial was conducted on 14 patients applying for the first time a high dose of 4200 mg BSE per day and 13 patients receiving placebo. For monitoring the serum levels of all major boswellic acids (BAs) a highly sensitive HPLC-MS method has been developed that allows the determination of KBA and AKBA from 5.0 ng/ml to 3000 ng/ml and of αBA, βBA, AαBA and AβBA from 0.5 ng/ml to 12,000 ng/ml. It is the first validated method that covers such a wide concentration range, which makes it suitable to be used as standard method in clinical trials as it compensates for the great pharmacokinetic variability in the plasma levels of BAs observed in clinical practice. Average steady concentrations (ng/ml) in the range of 6.4-247.5 for KBA, 0-15.5 for AKBA, 36.7-4830.1 for αBA, 87.0-11948.5 for βBA, 73.4-2985.8 for AαBA and 131.4-6131.3 for AβBA were determined in the verum group. The here quantified steady state levels suggest βBA to be a possible candidate for the anti-inflammatory and anti-edemateous effects of BSE. In general, the serum level analysis underlines the promising clinical results of BSE on cerebral edema.     

Toxicity and intracellular accumulation of bile acids in sandwich-cultured rat hepatocytes: Role of glycine conjugates

Excessive intrahepatic accumulation of bile acids (BAs) is a key mechanism underlying cholestasis. The aim of this study was to quantitatively explore the relationship between cytotoxicity of BAs and their intracellular accumulation in sandwich-cultured rat hepatocytes (SCRH). Following exposure of SCRH (on day-1 after seeding) to various BAs for 24 h, glycine-conjugated BAs were most potent in exerting toxicity. Moreover, unconjugated BAs showed significantly higher toxicity in day-1 compared to day-3 SCRH. When day-1/-3 SCRH were exposed (0.5–4 h) to 5–100 μM (C)DCA, intracellular levels of unconjugated (C)DCA were similar, while intracellular levels of glycine conjugates were up to 4-fold lower in day-3 compared to day-1 SCRH. Sinusoidal efflux was by far the predominant efflux pathway of conjugated BAs both in day-1 and day-3 SCRH, while canalicular BA efflux showed substantial interbatch variability. After 4 h exposure to (C)DCA, intracellular glycine conjugate levels were at least 10-fold higher than taurine conjugate levels. Taken together, reduced BA conjugate formation in day-3 SCRH results in lower intracellular glycine conjugate concentrations, explaining decreased toxicity of (C)DCA in day-3 versus day-1 SCRH. Our data provide for the first time a direct link between BA toxicity and glycine conjugate exposure in SCRH.     

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.     

Bridging cell surface receptor with nuclear receptors in control of bile acid homeostasis

Bile acids (BAs) are traditionally considered as “physiological detergents” for emulsifying hydrophobic lipids and vitamins due to their amphipathic nature. But accumulating clinical and experimental evidence shows an association between disrupted BA homeostasis and various liver disease conditions including hepatitis infection, diabetes and cancer. Consequently, BA homeostasis regulation has become a field of heavy interest and investigation. After identification of the Farnesoid X Receptor (FXR) as an endogenous receptor for BAs, several nuclear receptors (SHP, HNF4α, and LRH-1) were also found to be important in regulation of BA homeostasis. Some post-translational modifications of these nuclear receptors have been demonstrated, but their physiological significance is still elusive. Gut secrets FGF15/19 that can activate hepatic FGFR4 and its downstream signaling cascade, leading to repressed hepatic BA biosynthesis. However, the link between the activated kinases and these nuclear receptors is not fully elucidated. Here, we review the recent literature on signal crosstalk in BA homeostasis.     

Influence of the flavonoids apigenin, kaempferol, and quercetin on the function of organic anion transporting polypeptides 1A2 and 2B1

OATP1A2 and OATP2B1 are uptake transporters of the human organic anion transporting polypeptide (OATP) family with a broad substrate spectrum including several endogenous compounds as well as drugs such as the antihistaminic drug fexofenadine and HMG-CoA reductase inhibitors. Both transporters are localized in the apical membrane of human enterocytes. Flavonoids, abundantly occurring in plants, have previously been shown to interact with drug metabolizing enzymes and transporters. However, the impact of flavonoids on OATP1A2 and OATP2B1 transport function has not been analyzed in detail. Therefore, HEK293 cell lines stably expressing OATP1A2 and OATP2B1 were used to investigate the influence of the Ginkgo flavonoids apigenin, kaempferol, and quercetin on the transport activity of OATP1A2 and OATP2B1. K_i values of all three flavonoids determined from Dixon plot analyses using BSP as substrate indicated a competitive inhibition with quercetin as the most potent inhibitor of OATP1A2 (22.0 μM) and OATP2B1 (8.7 μM) followed by kaempferol (OATP1A2: 25.2 μM, OATP2B1: 15.1 μM) and apigenin (OATP1A2: 32.4 μM OATP2B1: 20.8 μM). Apigenin, kaempferol, and quercetin led to a concentration-dependent decrease of the OATP1A2-mediated fexofenadine transport with IC₅₀ values of 4.3 μM, 12.0 μM, and 12.6 μM, respectively. The OATP1A2- and OATP2B1-mediated transport of atorvastatin was also efficiently inhibited by apigenin (IC₅₀ for OATP1A2: 9.3 μM, OATP2B1: 13.9 μM), kaempferol (IC₅₀ for OATP1A2: 37.3 μM, OATP2B1: 20.7 μM) and quercetin (IC₅₀ for OATP1A2: 13.5 μM, OATP2B1: 14.1 μM). These data indicate that modification of OATP1A2 and OATP2B1 transport activity by apigenin, kaempferol, and quercetin may be a mechanism for food-drug or drug-drug interactions in humans.     

Expression of transporters potentially involved in the targeting of cytostatic bile acid derivatives to colon cancer and polyps

Drug targeting might help to overcome resistance to chemotherapy. Here we investigated whether colon cancer and polyps do express functional carriers involved in the uptake of cytostatic bile acid derivatives, in this case Bamet-UD2 [cis-diammine-bisursodeoxycholate-platinum(II)], which has been reported to be taken up by colon cancer cells "in vitro", efficiently induce apoptosis and overcome resistance to cisplatin. Although at lower levels than in ileum, a detectable expression of ASBT, OATP8/1B3, OCT1 and OSTalpha in colon tissue was found, which was not impaired in colon cancer or polyps. The expression of OATP-A/1A2 and OSTbeta was also found in colon, but this was markedly decreased in neoplastic colon tissue. In contrast, the expression of OATP-C/1B1 was low in colon but significantly enhanced in neoplastic colon tissue. MDR1 and MRP2 were poorly expressed in colon as compared with ileum, whereas MRP3 expression was higher in colon than in ileum. The abundance of mRNA for these ABC proteins was not changed in colon cancer or polyps. When RNA from different tissues was injected to Xenopus laevis oocytes their ability to take up taurocholate and Bamet-UD2 was enhanced (healthy ileum>healthy colon approximately neoplastic colon tissue). In all cases, uptake was lower for taurocholate than for Bamet-UD2, probably due to that ASBT mediates sodium-dependent uptake of both substrates, whereas additional transporters expressed in these tissues can participate in Bamet-UD2 uptake. In conclusion, our results suggest that the use of cytostatic bile acid derivatives might be a good pharmacological strategy for the treatment of colon tumors.     

Expression profiles of various transporters for oligopeptides, amino acids and organic ions along the human digestive tract

Various transporters such as H⁺/peptide cotransporter PEPT1 are expressed in the intestine, and play important physiological and pharmacological roles in the body. Present study was performed to examine the expression profile of 20 kinds of transporters (PEPT1 and 2, P-glycoprotein, amino acid transporters and organic ion transporters) along the human digestive tract, especially focusing on PEPT1. Using normal mucosal specimens, real-time polymerase chain reactions were carried out. Immunoblot analyses were also performed for PEPT1 expression. PEPT1 mRNA was highly expressed in the small intestine (duodenum > jejunum > ileum) compared to other tissues, and some patients showed a significant level of expression in the stomach. The expressional pattern of PEPT1 in the stomach and histological diagnosis indicated that gastric PEPT1 originated from the intestinal metaplasia. The amino acid transporters showed unique mRNA expression levels and distributions in the digestive tract. For example, the expression levels of B⁰AT1, a Na⁺-dependent and chloride-independent neutral amino acid transporter, were increased from the duodenum to ileum, which pattern is completely inverted to that for PEPT1. There is little expression of organic ion transporters except for organic cation/carnitine transporter OCTN2. In conclusion, PEPT1 was abundantly expressed in the small intestine, and the reciprocal expression of PEPT1 and B⁰AT1 may serve for the efficient absorption of protein digestive products.