The morphology, functionality, and longevity of a novel all human hepatic cell-based tri-culture system.

There remains a significant need for a convenient, phenotypically stable long-term culture platform for primary human hepatocytes (PHHs) for use in pharmacological and toxicological applications. Conventional in vitro models are often inconvenient, burdensome to use, and unable to support a multitude of donor lots or maintain PHH structural and functional properties over extended time. To address these limitations, an all-human cell-based hepatic tri-culture system (HTCS) has been developed comprised of frozen vials of PHHs and feeder cells. Qualified PHHs exhibited healthy morphological characteristics for ≥30 days. Extensive anastomosing networks of bile canaliculi with tight and gap junctions were established early and remained stable and functional throughout the culture period. After 5 culture days, albumin, urea, and basal Phase 1 and Phase 2 metabolic functions were stable for at least 2 weeks and significantly higher in the HTCS PHHs compared to sandwich monoculture PHHs. Induction of CYP functional activity by prototypical receptor agonists was stable after 4 days for at least 2 weeks. Gene expression of Alb and various CYPs in the HTCS PHHs was significantly higher compared to sandwich monoculture PHHs. The HTCS represents a convenient, phenotypically stable, all-human PHH culture platform for pharmacological and toxicological applications.

Effects of Overexpression of Fibroblast Growth Factor 15/19 on Hepatic Drug Metabolizing Enzymes.

Fibroblast growth factors 15 (FGF15) and 19 (FGF19) are endocrine growth factors that play an important role in maintaining bile acid homeostasis. FGF15/19-based therapies are currently being tested in clinical trials for the treatment of nonalcoholic steatohepatitis and cholestatic liver diseases. To determine the physiologic impact of long-term elevations of FGF15/19, a transgenic mouse model with overexpression of Fgf15 (Fgf15 Tg) was used in the current study. The RNA sequencing (RNA-seq) analysis revealed elevations of the expression of several genes encoding phase I drug metabolizing enzymes (DMEs), including Cyp2b10 and Cyp3a11, in Fgf15 Tg mice. We found that the induction of several Cyp2b isoforms resulted in increased function of CYP2B in microsomal metabolism and pharmacokinetics studies. Because the CYP2B family is known to be induced by constitutive androstane receptor (CAR), to determine the role of CAR in the observed inductions, we crossed Fgf15 Tg mice with CAR knockout mice and found that CAR played a minor role in the observed alterations in DME expression. Interestingly, we found that the overexpression of Fgf15 in male mice resulted in a phenotypical switch from the male hepatic expression pattern of DMEs to that of female mice. Differences in secretion of growth hormone (GH) between male and female mice are known to drive sexually dimorphic, STAT5b-dependent expression patterns of hepatic genes. We found that male Fgf15 Tg mice presented with many features similar to GH deficiency, including lowered body length and weight, Igf-1 and Igfals expression, and STAT5 signaling. SIGNIFICANCE STATEMENT: The overexpression of Fgf15 in mice causes an alteration in DMEs at the mRNA, protein, and functional levels, which is not entirely due to CAR activation but associated with lower GH signaling.

A framework using topological pathways for deeper analysis of transcriptome data.

BACKGROUND: Pathway analysis is one of the later stage data analysis steps essential in interpreting high-throughput gene expression data. We propose a set of algorithms which given gene expression data can recognize which portion of sub-pathways are actively utilized in the biological system being studied. The degree of activation is measured by conditional probability of the input expression data based on the Bayesian Network model constructed from the topological pathway. RESULTS: We demonstrate the effectiveness of our pathway analysis method by conducting two case studies. The first one applies our method to a well-studied temporal microarray data set for the cell cycle using the KEGG Cell Cycle pathway. Our method closely reproduces the biological claims associated with the data sets, but unlike the original work ours can produce how pathway routes interact with each other above and beyond merely identifying which pathway routes are involved in the process. The second study applies the method to the p53 mutation microarray data to perform a comparative study. CONCLUSIONS: We show that our method achieves comparable performance against all other pathway analysis systems included in this study in identifying p53 altered pathways. Our method could pave a new way of carrying out next generation pathway analysis.

BioTarget: A Computational Framework Identifying Cancer Type Specific Transcriptional Targets of Immune Response Pathways.

Transcriptome data can provide information on signaling pathways active in cancers, but new computational tools are needed to more accurately quantify pathway activity and identify tissue-specific pathway features. We developed a computational method called "BioTarget" that incorporates ChIP-seq data into cellular pathway analysis. This tool relates the expression of transcription factor TF target genes (based on ChIP-seq data) with the status of upstream signaling components for an accurate quantification of pathway activity. This analysis also reveals TF targets expressed in specific contexts/tissues. We applied BioTarget to assess the activity of TBX21 and GATA3 pathways in cancers. TBX21 and GATA3 are TF regulators that control the differentiation of T cells into Th1 and Th2 helper cells that mediate cell-based and humoral immune responses, respectively. Since tumor immune responses can impact cancer progression, the significance of our pathway scores should be revealed by effective patient stratification. We found that low Th1/Th2 activity ratios were associated with a significantly poorer survival of stomach and breast cancer patients, whereas an unbalanced Th1/Th2 response was correlated with poorer survival of colon cancer patients. Lung adenocarcinoma and lung squamous cell carcinoma patients had the lowest survival rates when both Th1 and Th2 responses were high. Our method also identified context-specific target genes for TBX21 and GATA3. Applying the BioTarget tool to BCL6, a TF associated with germinal center lymphocytes, we observed that patients with an active BCL6 pathway had significantly improved survival for breast, colon, and stomach cancer. Our findings support the effectiveness of the BioTarget tool for transcriptome analysis and point to interesting associations between some immune-response pathways and cancer progression.

Consequences of Phenytoin Exposure on Hepatic Cytochrome P450 Expression during Postnatal Liver Maturation in Mice.

The induction of cytochrome P450 (P450) enzymes in response to drug treatment is a significant contributing factor to drug-drug interactions, which may reduce therapeutic efficacy and/or cause toxicity. Since most studies on P450 induction are performed in adults, enzyme induction at neonatal, infant, and adolescent ages is not well understood. Previous work defined the postnatal ontogeny of drug-metabolizing P450s in human and mouse livers; however, there are limited data on the ontogeny of the induction potential of each enzyme in response to drug treatment. Induction of P450s at the neonatal age may also cause permanent alterations in P450 expression in adults. The goal of this study was to investigate the short- and long-term effects of phenytoin treatment on mRNA and protein expressions and enzyme activities of CYP2B10, 2C29, 3A11, and 3A16 at different ages during postnatal liver maturation in mice. Induction of mRNA immediately following phenytoin treatment appeared to depend on basal expression of the enzyme at a specific age. While neonatal mice showed the greatest fold changes in CYP2B10, 2C29, and 3A11 mRNA expression following treatment, the levels of induced protein expression and enzymatic activity were much lower than that of induced levels in adults. The expression of fetal CYP3A16 was repressed by phenytoin treatment. Neonatal treatment with phenytoin did not permanently induce enzyme expression in adulthood. Taken together, our data suggest that inducibility of drug-metabolizing P450s is much lower in neonatal mice than it is in adults and neonatal induction by phenytoin is not permanent.

A Transcriptional Regulatory Network Containing Nuclear Receptors and Long Noncoding RNAs Controls Basal and Drug-Induced Expression of Cytochrome P450s in HepaRG Cells.

Cytochrome P450 (P450) enzymes are responsible for metabolizing drugs. Expression of P450s can directly affect drug metabolism, resulting in various outcomes in therapeutic efficacy and adverse effects. Several nuclear receptors are transcription factors that can regulate expression of P450s at both basal and drug-induced levels. Some long noncoding RNAs (lncRNAs) near a transcription factor are found to participate in the regulatory functions of the transcription factors. The aim of this study is to determine whether there is a transcriptional regulatory network containing nuclear receptors and lncRNAs controlling both basal and drug-induced expression of P450s in HepaRG cells. Small interfering RNAs or small hairpin RNAs were applied to knock down four nuclear receptors [hepatocyte nuclear factor 1α (HNF1α), hepatocyte nuclear factor 4α (HNF4α), pregnane X receptor (PXR), and constitutive androstane receptor (CAR)] as well as two lncRNAs [HNF1α antisense RNA 1 (HNF1α-AS1) and HNF4α antisense RNA 1 (HNF4α-AS1)] in HepaRG cells with or without treatment of phenobarbital or rifampicin. Expression of eight P450 enzymes was examined in both basal and drug-induced levels. CAR and PXR mainly regulated expression of specific P450s. HNF1α and HNF4α affected expression of a wide range of P450s as well as other transcription factors. HNF1α and HNF4α controlled the expression of their neighborhood lncRNAs, HNF1α-AS1 and HNF4α-AS1, respectively. HNF1α-AS1 and HNF4α-AS1 was also involved in the regulation of P450s and transcription factors in diverse manners. Altogether, our study concludes that a transcription regulatory network containing the nuclear receptors and lncRNAs controls both basal and drug-induced expression of P450s in HepaRG cells.

The role of H19, a long non-coding RNA, in mouse liver postnatal maturation.

H19 RNA is highly expressed at early postnatal ages and precipitously decreases at a specific time corresponding with increases in expression of genes important for mature liver function, such as drug metabolizing enzymes. H19's role in the regulation of liver maturation is currently unknown. Using an H19 knockout mouse model to determine the role of H19 in liver development, we quantified gene expression for insulin growth factor signaling, Wnt signaling, key cytochrome P450 (P450) enzymes known to change as the liver develops, and fetal and adult plasma protein produced in liver. In mice lacking H19 expression, liver weights were significantly increased immediately after birth and significant increases were found in the number of actively proliferating cells. Increases in cell proliferation may be due to increases in ß-catenin protein affecting Wnt signaling, increases in insulin-like growth factor 2 (IGF2) expression, and/or increases in insulin-like growth factor 1 receptor (IGF1R) expression at the protein level. Loss of targeted inhibition of IGF1R by microRNA 675 (miR-675) may be the cause of IGF1R increases, as miR-675 expression is also abrogated with loss of H19 expression in our model. P450 expression patterns were largely unchanged. No change in the production of plasma proteins was found, indicating H19 may not be important for liver maturation despite its role in controlling cell proliferation during liver growth. H19 may be important for normal liver development, and understanding how the liver matures will assist in predicting drug efficacy and toxicity in pediatric populations.

Impact of Drug Treatment at Neonatal Ages on Variability of Drug Metabolism and Drug-drug Interactions in Adult Life.

PURPOSE OF REVIEW: As the number of patients taking more than one medication concurrently continues to increase, predicting and preventing drug-drug interactions (DDIs) is now more important than ever. Administration of one drug can cause changes in the expression and activity of drug metabolizing enzymes (DMEs) and alter the efficacy or toxicity of other medications that are substrates for these enzymes, resulting in a DDI. In today's medical practice, potential DDIs are evaluated based on the current medications a patient is taking with little regard to drugs the patient has been exposed to in the past. The purpose of this review is to discuss potential impacts of drug treatment at neonatal ages on the variability of drug metabolism and DDIs in adult life. RECENT FINDINGS: Existing evidence from the last thirty years has shown that exposure to certain xenobiotics during neonatal life has the potential to persistently alter DME expression through adult life. With recent advancements in the understanding of epigenetic regulation on gene expression, this phenomenon is resurfacing in the scientific community in hopes of defining possible mechanisms. Exposure to compounds that have the ability to bind nuclear receptors and trigger epigenetic modifications at neonatal and pediatric ages may have long-term, if not permanent, consequences on gene expression and DME activity. SUMMARY: The information summarized in this review should challenge the way current healthcare providers assess DDI potential and may offer an explanation to the significant interindividual variability in drug metabolism that is observed among patients.

Phenobarbital Treatment at a Neonatal Age Results in Decreased Efficacy of Omeprazole in Adult Mice.

Drug-drug interactions (DDIs) occur when the action of one drug interferes with or alters the activity of another drug taken concomitantly. This can lead to decreased drug efficacy or increased toxicity. Because of DDIs, physicians in the clinical practice attempt to avoid potential interactions when multiple drugs are coadministrated; however, there is still a large knowledge gap in understanding how drugs taken in the past can contribute to DDIs in the future. The goal of this study was to investigate the consequence of neonatal drug exposure on efficacy of other drugs administered up through adult life. We selected a mouse model to test phenobarbital exposure at a neonatal age and its impact on efficacy of omeprazole in adult life. The results of our experiment show an observed decrease in omeprazole's ability to raise gastric pH in adult mice that received single or multiple doses of phenobarbital at a neonatal age. This effect may be associated with the permanent induction of cytochrome P450 enzymes in adult liver after neonatal phenobarbital treatment. Our data indicates that DDIs may result from drugs administered in the past in an animal model and should prompt re-evaluation of how DDIs are viewed and how to avoid long-term DDIs in clinical practice.

Role of Farnesoid X Receptor in the Determination of Liver Transcriptome during Postnatal Maturation in Mice.

The liver is a vital organ with critical functions in metabolism of various biologically useful materials, synthesis of several vital proteins, detoxification of toxic substances, and immune defense. Most liver functions are not mature at birth and many changes happen during postnatal liver development, which lead to differential vulnerabilities of the liver at different developmental stages. However, the details of what changes occur in liver after birth, at what developmental stages they occur, and molecular mechanisms in the regulation of the developmental process are not clearly known. The nuclear receptor Farnesoid X receptor (FXR) is an important transcriptional regulator in liver. Here, we used RNA-Sequencing to analyze the transcriptome of mouse liver from perinatal to adult ages in both C57BL/6 and Fxr-/- mice. We have defined a clear timeline of functional transition from prenatal through neonatal and adolescent to adult in C57BL/6 mice. Without FXR, activation of neonatal-specific pathways was prolonged and maturation of multiple metabolic pathways was delayed. The loss of FXR also led to increased expression of 27 other transcription regulators. Our data support a conclusion that developmental transcriptome revealed significant functional transition during postnatal liver development and FXR plays an important role in control of postnatal liver maturation.

Role of farnesoid X receptor in establishment of ontogeny of phase-I drug metabolizing enzyme genes in mouse liver.

The expression of phase-I drug metabolizing enzymes in liver changes dramatically during postnatal liver maturation. Farnesoid X receptor (FXR) is critical for bile acid and lipid homeostasis in liver. However, the role of FXR in regulating ontogeny of phase-I drug metabolizing genes is not clear. Hence, we applied RNA-sequencing to quantify the developmental expression of phase-I genes in both Fxr-null and control (C57BL/6) mouse livers during development. Liver samples of male C57BL/6 and Fxr-null mice at 6 different ages from prenatal to adult were used. The Fxr-null showed an overall effect to diminish the "day-1 surge" of phase-I gene expression, including cytochrome P450s at neonatal ages. Among the 185 phase-I genes from 12 different families, 136 were expressed, and differential expression during development occurred in genes from all 12 phase-I families, including hydrolysis: carboxylesterase (Ces), paraoxonase (Pon), and epoxide hydrolase (Ephx); reduction: aldoketo reductase (Akr), quinone oxidoreductase (Nqo), and dihydropyrimidine dehydrogenase (Dpyd); and oxidation: alcohol dehydrogenase (Adh), aldehyde dehydrogenase (Aldh), flavin monooxygenases (Fmo), molybdenum hydroxylase (Aox and Xdh), cytochrome P450 (P450), and cytochrome P450 oxidoreductase (Por). The data also suggested new phase-I genes potentially targeted by FXR. These results revealed an important role of FXR in regulation of ontogeny of phase-I genes.

Developmental programming of long non-coding RNAs during postnatal liver maturation in mice.

The liver is a vital organ with critical functions in metabolism, protein synthesis, and immune defense. Most of the liver functions are not mature at birth and many changes happen during postnatal liver development. However, it is unclear what changes occur in liver after birth, at what developmental stages they occur, and how the developmental processes are regulated. Long non-coding RNAs (lncRNAs) are involved in organ development and cell differentiation. Here, we analyzed the transcriptome of lncRNAs in mouse liver from perinatal (day -2) to adult (day 60) by RNA-Sequencing, with an attempt to understand the role of lncRNAs in liver maturation. We found around 15,000 genes expressed, including about 2,000 lncRNAs. Most lncRNAs were expressed at a lower level than coding RNAs. Both coding RNAs and lncRNAs displayed three major ontogenic patterns: enriched at neonatal, adolescent, or adult stages. Neighboring coding and non-coding RNAs showed the trend to exhibit highly correlated ontogenic expression patterns. Gene ontology (GO) analysis revealed that some lncRNAs enriched at neonatal ages have their neighbor protein coding genes also enriched at neonatal ages and associated with cell proliferation, immune activation related processes, tissue organization pathways, and hematopoiesis; other lncRNAs enriched at adolescent ages have their neighbor protein coding genes associated with different metabolic processes. These data reveal significant functional transition during postnatal liver development and imply the potential importance of lncRNAs in liver maturation.