The pregnane X receptor (PXR) is a ligand-activated nuclear receptor controlling hepatocyte expression of numerous genes. Although expression changes in xenobiotic-metabolizing, lipogenic, gluconeogenic and bile acid synthetic genes have been described after PXR activation, the temporal dynamics of their expression is largely unknown. Recently, 3D spheroids of primary human hepatocytes (PHHs) have been characterized as the most phenotypically relevant hepatocyte model. We used 3D PHHs to assess time-dependent expression profiles of 12 prototypic PXR-controlled genes in the time course of 168 h of rifampicin treatment (1 or 10 μM). We observed a similar bell-shaped time-induction pattern for xenobiotic-handling genes (CYP3A4, CYP2C9, CYP2B6, and MDR1). However, we observed either biphasic profiles for genes involved in endogenous metabolism (FASN, GLUT2, G6PC, PCK1, and CYP7A1), a decrease for SHP or oscillation for PDK4 and PXR. The rifampicin concentration determined the expression profiles for some genes. Moreover, we calculated half-lives of CYP3A4 and CYP2C9 mRNA under induced or basal conditions and we used a mathematical model to describe PXR-mediated regulation of CYP3A4 expression employing 3D PHHs. The study shows the importance of long-term time-expression profiling of PXR target genes in phenotypically stable 3D PHHs and provides insight into PXR function in liver beyond our knowledge from conventional 2D in vitro models.
Prediction methods have become an integral part of biomedical and biotechnological research. However, their clinical interpretations are largely based on biochemical or molecular data, but not clinical data. Here, we focus on improving the reliability and clinical applicability of prediction algorithms. We assembled and curated two large non-overlapping large databases of clinical phenotypes. These phenotypes were caused by missense variations in 44 and 63 genes associated with Mendelian diseases. We used these databases to establish and validate the model, allowing us to improve the predictions obtained from EVmutation, SNAP2 and PoPMuSiC 2.1. The predictions of clinical effects suffered from a lack of specificity, which appears to be the common constraint of all recently used prediction methods, although predictions mediated by these methods are associated with nearly absolute sensitivity. We introduced evidence-based tailoring of the default settings of the prediction methods; this tailoring substantially improved the prediction outcomes. Additionally, the comparisons of the clinically observed and theoretical variations led to the identification of large previously unreported pools of variations that were under negative selection during molecular evolution. The evolutionary variation analysis approach described here is the first to enable the highly specific identification of likely disease-causing missense variations that have not yet been associated with any clinical phenotype.
- MeSH
- Algorithms MeSH
- Ectodysplasins genetics MeSH
- Phenotype MeSH
- Genetic Variation MeSH
- Genetic Diseases, Inborn genetics MeSH
- Genomics MeSH
- Glucosephosphate Dehydrogenase genetics MeSH
- Hemoglobins genetics MeSH
- Hepatocyte Nuclear Factor 4 genetics MeSH
- Humans MeSH
- Mutation, Missense MeSH
- Models, Genetic * MeSH
- Evolution, Molecular MeSH
- Mutation * MeSH
- Likelihood Functions MeSH
- Proteomics MeSH
- Protein Tyrosine Phosphatase, Non-Receptor Type 11 genetics MeSH
- Computational Biology methods MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND & AIMS: Parenteral nutrition (PN), a lifesaving therapy in patients with intestinal failure, has been associated with hepatobiliary complications including steatosis, cholestasis and fibrosis, collectively known as parenteral nutrition-associated liver disease (PNALD). To date, the pathogenesis of PNALD is poorly understood and therapeutic options are limited. Impaired bile salt homeostasis has been proposed to contribute PNALD. The objective of this study was to establish a PNALD model in rats and to evaluate the effects of continuous parenteral nutrition (PN) on bile salt homeostasis. METHODS: Rats received either PN via the jugular vein or received normal diet for 3, 7 or 14 days. Serum biochemistry, hepatic triglycerides, circulating bile salts and C4, IL-6 and TNF-alpha, and lipogenic and bile salt homeostatic gene expression in liver and ileum were assessed. RESULTS: PN increased hepatic triglycerides already after 3 days of administration, and resulted in conjugated bilirubin elevation after 7 or more days. This indicates PN-induced steatosis and impaired canalicular secretion of bilirubin, the latter which is in line with reduced hepatic expression of Mrp2 mRNA. There was no histological evidence for liver inflammation after PN administration, and circulating levels of pro-inflammatory cytokines IL-6 and TNF-α, were comparable in all groups. Hepatic expression of Fxr mRNA was decreased after 7 days of PN, without apparent effect on expression of Fxr targets Bsep and Shp. Nonetheless, Cyp7a1 expression was reduced after 7 days of PN, indicative for lowered bile salt synthesis. Circulating levels of C4 (marker of bile salt synthesis) were also decreased after 3, 7 and 14 days of PN. Levels of circulating bile salts were not affected by PN. CONCLUSIONS: This study showed that PN in rats caused early mild steatosis and cholestasis, while hepatic and systemic inflammation were not present. The onset of these abnormalities was associated with alterations in bile salt synthesis and transport. This animal model serves as an experimental model to further investigate the pathogenesis of PNALD inflicted by steatosis and cholestasis.
- MeSH
- Alanine Transaminase blood MeSH
- Alkaline Phosphatase blood MeSH
- Aspartate Aminotransferases blood MeSH
- Bilirubin blood MeSH
- Cytokines blood MeSH
- gamma-Glutamyltransferase blood MeSH
- Homeostasis * MeSH
- Hyperbilirubinemia blood etiology MeSH
- Hypoalbuminemia blood etiology MeSH
- Liver metabolism physiopathology MeSH
- Rats MeSH
- Disease Models, Animal MeSH
- Liver Diseases blood etiology MeSH
- Intestinal Diseases therapy MeSH
- Parenteral Nutrition adverse effects MeSH
- Rats, Sprague-Dawley MeSH
- Triglycerides blood MeSH
- Bile Acids and Salts blood MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Particularly in the pediatric clinical pharmacology field, data-sharing offers the possibility of making the most of all available data. In this study, we utilize previously collected therapeutic drug monitoring (TDM) data of term and preterm newborns to develop a population pharmacokinetic model for phenobarbital. We externally validate the model using prospective phenobarbital data from an ongoing pharmacokinetic study in preterm neonates. METHODS: TDM data from 53 neonates (gestational age (GA): 37 (24-42) weeks, bodyweight: 2.7 (0.45-4.5) kg; postnatal age (PNA): 4.5 (0-22) days) contained information on dosage histories, concentration and covariate data (including birth weight, actual weight, post-natal age (PNA), postmenstrual age, GA, sex, liver and kidney function, APGAR-score). Model development was carried out using NONMEM® 7.3. After assessment of model fit, the model was validated using data of 17 neonates included in the DINO (Drug dosage Improvement in NeOnates)-study. RESULTS: Modelling of 229 plasma concentrations, ranging from 3.2 to 75.2mg/L, resulted in a one compartment model for phenobarbital. Clearance (CL) and volume (Vd) for a child with a birthweight of 2.6kg at PNA day 4.5 was 0.0091L/h (9%) and 2.38L (5%), respectively. Birthweight and PNA were the best predictors for CL maturation, increasing CL by 36.7% per kg birthweight and 5.3% per postnatal day of living, respectively. The best predictor for the increase in Vd was actual bodyweight (0.31L/kg). External validation showed that the model can adequately predict the pharmacokinetics in a prospective study. CONCLUSION: Data-sharing can help to successfully develop and validate population pharmacokinetic models in neonates. From the results it seems that both PNA and bodyweight are required to guide dosing of phenobarbital in term and preterm neonates.
- MeSH
- Phenobarbital administration & dosage MeSH
- Infant MeSH
- Humans MeSH
- Drug Monitoring methods MeSH
- Infant, Premature MeSH
- Infant, Newborn MeSH
- Prospective Studies MeSH
- Information Dissemination methods MeSH
- Dose-Response Relationship, Drug MeSH
- Check Tag
- Infant MeSH
- Humans MeSH
- Male MeSH
- Infant, Newborn MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Epigallocatechin gallate (EGCG) has been shown to be protective in various experimental models of liver injury, although opposite effects have also been reported. Since its effect on biliary physiology has not been thoroughly investigated, the present study evaluated effect of EGCG on bile flow and bile acid homeostasis in rats. Compared to controls, EGCG treatment decreased bile flow by 23%. Hepatic paracellular permeability and biliary bile acid excretion were not altered by EGCG administration, but biliary glutathione excretion was reduced by 70%. Accordingly, the main glutathione transporter on the hepatocyte canalicular membrane, multidrug resistance-associated protein 2 (Mrp2), was significantly decreased at the protein level. EGCG administration also doubled plasma bile acid levels compared to controls. While protein levels of the main hepatic bile acid transporters were unchanged, the rate-limiting enzyme in the bile acid synthesis, Cyp7a1, was significantly increased by EGCG. Enhanced bile acid synthesis in these animals was also confirmed by a 2-fold increase in plasma marker 7α-hydroxy-4-cholesten-3-one. In contrast, EGCG markedly downregulated major bile acid transporters (Asbt and Ostα) and regulatory molecules (Shp and Fgf15) in the ileum. When EGCG was coadministered with ethinylestradiol, a potent cholestatic agent, it did not show any additional effect on the induced cholestasis. This study shows ability of EGCG to raise plasma bile acid concentrations, mainly through Cyp7a1 upregulation, and to decrease bile production through reduction in Mrp2-mediated bile acid-independent bile flow. In conclusion, our data demonstrate that under certain conditions EGCG may induce cholestasis.
- MeSH
- ATP-Binding Cassette Transporters genetics MeSH
- Cholestasis chemically induced MeSH
- Cholestenones metabolism MeSH
- Cholesterol 7-alpha-Hydroxylase genetics metabolism MeSH
- Down-Regulation drug effects MeSH
- Ethinyl Estradiol pharmacology MeSH
- Glutathione metabolism MeSH
- Hepatocytes drug effects metabolism MeSH
- Homeostasis drug effects MeSH
- Ileum drug effects metabolism MeSH
- Catechin analogs & derivatives toxicity MeSH
- Rats MeSH
- Permeability MeSH
- Rats, Wistar MeSH
- Up-Regulation drug effects MeSH
- Bile Acids and Salts biosynthesis metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The aim of this study was to investigate the nycthemeral rhythm of total locomotor activity (TLA) in horse and the possible involvement of the daily organization of rest/activity cycles in the fluctuation of the redox state. For this purpose we recorded TLA and determined oxidative markers in ten clinically healthy Italian Saddle horses. TLA was continuously recorded by means of an actigraphy-based data logger Actiwatch-Mini®. For the assessment of free radicals (dROMs), the antioxidant barrier (Oxy-ads) and the thiol-antioxydant barrier (SHp), blood samples were collected every 4 hours over a 48 h period. One-way repeated measures analysis of variance (ANOVA) showed a statistically significant effect of time of day on all studied parameters. The application of the periodic model and the statistical analysis of cosinor indicate, in horses, the existence of a daily rhythm of the studied parameters during the 48 h of monitoring of the horses. The results show that nycthemeral rhythms of TLA and oxidative markers have different trends in horse. dROMs and Oxy-ads showed a nycthemeral rhythm with an acrophase in the middle of the photophase, and an acrophase of SHp nycthemeral rhythm preceded them. In contrast, TLA showed its acrophase only after the middle of the photophase. TLA showed a lower robustness of rhythms (16.3 and 20.3%) and in respect to the robustness values of the rhythms of oxidative markers (67.3-86.2%). In conclusion, the results of the present investigation showed that oxidative markers have different patterns than locomotor activity, and further studies could be necessary to determine whether other external stimuli, such as solar radiation, food administration or physical exercise are able to influence redox state rhythms in this species.
- MeSH
- Endothelial Growth Factors physiology MeSH
- Ischemia diagnosis physiopathology therapy MeSH
- Extremities pathology MeSH
- Rats MeSH
- Angiogenesis Inducing Agents MeSH
- Disease Models, Animal MeSH
- Transduction, Genetic methods MeSH
- Tyrosine physiology metabolism MeSH
- Treatment Outcome MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
