Unnatural Endotype B PPAPs as Novel Compounds with Activity against Mycobacterium tuberculosis.

Pre-SARS-CoV-2, tuberculosis was the leading cause of death by a single pathogen. Repetitive exposure of Mycobacterium tuberculosis(Mtb) supported the development of multidrug- and extensively drug-resistant strains, demanding novel drugs. Hyperforin, a natural type A polyprenylated polycyclic acylphloroglucinol from St. John's wort, exhibits antidepressant and antibacterial effects also against Mtb. Yet, Hyperforin's instability limits the utility in clinical practice. Here, we present photo- and bench-stable type B PPAPs with enhanced antimycobacterial efficacy. PPAP22 emerged as a lead compound, further improved as the sodium salt PPAP53, drastically enhancing solubility. PPAP53 inhibits the growth of virulent extracellular and intracellular Mtb without harming primary human macrophages. Importantly, PPAP53 is active against drug-resistant strains of Mtb. Furthermore, we analyzed the in vitro properties of PPAP53 in terms of CYP induction and the PXR interaction. Taken together, we introduce type PPAPs as a new class of antimycobacterial compounds, with remarkable antibacterial activity and favorable biophysical properties.

Effects of Diminished NADPH:cytochrome P450 Reductase in Human Hepatocytes on Lipid and Bile Acid Homeostasis.

NADPH:cytochrome P450 oxidoreductase (POR) is the obligate electron donor for microsomal cytochrome P450 (CYP) enzymes involved in the biosynthesis of endogenous substances like bile acids and other steroids as well as in the oxidative metabolism of xenobiotics. P450 oxidoreductase also supports other redox enzymes in fatty acid and cholesterol pathways. Recently, we have established CRISPR/Cas9-mediated POR knockdown in a human hepatic cell model, HepaRG, and demonstrated the differential effects of limited POR expression on CYP activity. The aim of the present work was to systematically investigate the impact of POR knockdown with a focus on the expression of ADME (absorption, distribution, metabolism, and excretion) genes and related regulators. Functional consequences have been assessed using quantitative mass spectrometry for targeted metabolomics covering bile acids, and cholesterol and its precursors, and for untargeted proteomics. In addition to the previously described alteration of RNA expression of CYP genes, we showed significant downregulation of transcriptional regulators of drug metabolism and transport, including NR1I3 (CAR), NR1I2 (PXR), NR1H4 (FXR), and NR1H3 (LXRα) in cells with POR gene disruption. Furthermore, POR knockdown resulted in deregulated bile acid and cholesterol biosynthesis demonstrated by low levels of cholic acid derivates and increased concentrations of chenodeoxycholic acid derivates, respectively. Systemic effects of POR knockdown on global protein expression were indicated by downregulation of several metabolic pathways including lipid metabolism and biological oxidation reactions. The deduced protein network map corroborates CYP enzymes as direct interaction partners, whereas changes in lipid metabolism and homeostasis are the result of indirect effects. In summary, our results emphasize a widespread role of POR in various metabolic pathways and provide the first human data on the effects of diminished POR expression on drug and endogenous metabolism in a genomeedited HepaRG cell model.

Differential effects on human cytochromes P450 by CRISPR/Cas9-induced genetic knockout of cytochrome P450 reductase and cytochrome b5 in HepaRG cells.

HepaRG cells are increasingly accepted as model for human drug metabolism and other hepatic functions. We used lentiviral transduction of undifferentiated HepaRG cells to deliver Cas9 and two alternative sgRNAs targeted at NADPH:cytochrome P450 oxidoreductase (POR), the obligate electron donor for microsomal cytochromes P450 (CYP). Cas9-expressing HepaRGVC (vector control) cells were phenotypically similar to wild type HepaRG cells and could be differentiated into hepatocyte-like cells by DMSO. Genetic POR-knockout resulted in phenotypic POR knockdown of up to 90% at mRNA, protein, and activity levels. LC-MS/MS measurement of seven CYP-activities showed differential effects of POR-knockdown with CYP2C8 being least and CYP2C9 being most affected. Further studies on cytochrome b5 (CYB5), an alternative NADH-dependent electron donor indicated particularly strong support of CYP2C8-dependent amodiaquine N-deethylation by CYB5 and this was confirmed by genetic CYB5 single- and POR/CYB5 double-knockout. POR-knockdown also affected CYP expression on mRNA and protein level, with CYP1A2 being induced severalfold, while CYP2C9 was strongly downregulated. In summary our results show that POR/NADPH- and CYB5/NADH-electron transport systems influence human drug metabolizing CYPs differentially and differently than mouse Cyps. Our Cas9-expressing HepaRGVC cells should be suitable to study the influence of diverse genes on drug metabolism and other hepatic functions.

Tri-Allelic Haplotypes Determine and Differentiate Functionally Normal Allele CYP2D6*2 and Impaired Allele CYP2D6*41.

CYP2D6 metabolizes 20-25% of all clinically used drugs and its complex genetic polymorphism is a major determinant of drug safety and efficacy. We investigated the basis for the functional difference between the two common alleles *2 (g.2851C>T + g.4181G>C, normal function) and *41 (additional intronic g.2989G>A, reduced function). A recently reported far-distant enhancer polymorphism rs5758550A/G linked to *2 has been suggested to play a decisive role. Genotyping of two white cohorts confirmed strong linkage of rs5758550G to *2, whereas no influence was found on metabolic ratio of sparteine or hepatic expression. Genomic plasmid constructs carrying individual variants or combinations thereof were expressed in COS1 and Huh7 cells. Both g.2851C>T(R296C) and g.2989G>A reduced enzyme activity and protein levels similarly by ~ 50-65% compared to reference (*1), whereas the double variant had only ~ 20% activity. Although the unexpected loss of function caused by g.2851C>T was compensated by g.4181G>C (mimicking the EM-phenotype of *2), the additional loss of function due to intronic g.2989G>A in the triple variant was not compensated (mimicking the IM-phenotype of *41). We also confirmed increased erroneous splicing in carriers of g.2989G>A but not of g.2851C>T as a likely explanation for the impaired function of *41. In conclusion, our data demonstrate g.2989G>A as causal variant of impaired allele CYP2D6*41 whereas triple-haplotypes have to be considered to explain the functional difference between *2 and *41. These data are important for genotyping strategies and clinical implementation of CYP2D6 pharmacogenetics.

Progesterone receptor membrane component 1 regulates lipid homeostasis and drives oncogenic signaling resulting in breast cancer progression.

BACKGROUND: PGRMC1 (progesterone receptor membrane component 1) is a highly conserved heme binding protein, which is overexpressed especially in hormone receptor-positive breast cancer and plays an important role in breast carcinogenesis. Nevertheless, little is known about the mechanisms by which PGRMC1 drives tumor progression. The aim of our study was to investigate the involvement of PGRMC1 in cholesterol metabolism to detect new mechanisms by which PGRMC1 can increase lipid metabolism and alter cancer-related signaling pathways leading to breast cancer progression. METHODS: The effect of PGRMC1 overexpression and silencing on cellular proliferation was examined in vitro and in a xenograft mouse model. Next, we investigated the interaction of PGRMC1 with enzymes involved in the cholesterol synthesis pathway such as CYP51, FDFT1, and SCD1. Further, the impact of PGRMC1 expression on lipid levels and expression of enzymes involved in lipid homeostasis was examined. Additionally, we assessed the role of PGRMC1 in key cancer-related signaling pathways including EGFR/HER2 and ERα signaling. RESULTS: Overexpression of PGRMC1 resulted in significantly enhanced proliferation. PGRMC1 interacted with key enzymes of the cholesterol synthesis pathway, alters the expression of proteins, and results in increased lipid levels. PGRMC1 also influenced lipid raft formation leading to altered expression of growth receptors in membranes of breast cancer cells. Analysis of activation of proteins revealed facilitated ERα and EGFR activation and downstream signaling dependent on PGRMC1 overexpression in hormone receptor-positive breast cancer cells. Depletion of cholesterol and fatty acids induced by statins reversed this growth benefit. CONCLUSION: PGRMC1 may mediate proliferation and progression of breast cancer cells potentially by altering lipid metabolism and by activating key oncogenic signaling pathways, such as ERα expression and activation, as well as EGFR signaling. Our present study underlines the potential of PGRMC1 as a target for anti-cancer therapy.

TGF-ß2 silencing to target biliary-derived liver diseases.

OBJECTIVE: TGF-ß2 (TGF-ß, transforming growth factor beta), the less-investigated sibling of TGF-ß1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-ß2 in biliary-derived liver diseases. DESIGN: As we also found upregulated TGFB2 in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-ß2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of TgfB2 silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels. RESULTS: TgfB2-induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. TgfB2 expression in MDR2-KO mice was blunted using TgfB2-directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced PparG expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on Ccl3, Ccl4, Ccl5, Mki67 and Notch3 expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, TGFB2 and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue. CONCLUSIONS: Taken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-ß2 silencing and provide a direct rationale for TGF-ß2-directed drug development.

Polycyclic Aromatic Hydrocarbons Activate the Aryl Hydrocarbon Receptor and the Constitutive Androstane Receptor to Regulate Xenobiotic Metabolism in Human Liver Cells.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants produced by incomplete combustion of organic matter. They induce their own metabolism by upregulating xenobiotic-metabolizing enzymes such as cytochrome P450 monooxygenase 1A1 (CYP1A1) by activating the aryl hydrocarbon receptor (AHR). However, previous studies showed that individual PAHs may also interact with the constitutive androstane receptor (CAR). Here, we studied ten PAHs, different in carcinogenicity classification, for their potential to activate AHR- and CAR-dependent luciferase reporter genes in human liver cells. The majority of investigated PAHs activated AHR, while non-carcinogenic PAHs tended to activate CAR. We further characterized gene expression, protein abundancies and activities of the AHR targets CYP1A1 and 1A2, and the CAR target CYP2B6 in human HepaRG hepatoma cells. Enzyme induction patterns strongly resembled the profiles obtained at the receptor level, with AHR-activating PAHs inducing CYP1A1/1A2 and CAR-activating PAHs inducing CYP2B6. In summary, this study provides evidence that beside well-known activation of AHR, some PAHs also activate CAR, followed by subsequent expression of respective target genes. Furthermore, we found that an increased PAH ring number is associated with AHR activation as well as the induction of DNA double-strand breaks, whereas smaller PAHs activated CAR but showed no DNA-damaging potential.

Copy number variation profiling in pharmacogenes using panel-based exome resequencing and correlation to human liver expression.

Structural variants including copy number variations (CNV) have gained widespread attention, especially in pharmacogenomics but for several genes functional relevance and clinical evidence are still lacking. Detection of CNVs in next-generation sequencing data is challenging but offers widespread applications. We developed a cohort-based CNV detection workflow to extract CNVs from read counts of targeted NGS of 340 genes involved in absorption, distribution, metabolism and excretion (ADME) of drugs. We applied our method to 150 human liver tissue samples and correlated identified CNVs to mRNA expression levels. In total, we identified 445 deletions (73%) and 167 duplications (27%) in 36 pharmacogenes including all well-known CNVs of CYPs, GSTs, SULTs, UGTs, numerous described rare CNVs of CYP2E1, SLC16A3 or UGT2B15 as well as novel observations, e.g., for SLC22A12, SLC22A17 and GPS2 (G Protein Pathway Suppressor 2). We were able to fine-map complex CNVs of CYP2A6 and CYP2D6 with exon resolution. Correlation analysis confirmed known expression patterns for common CNVs and suggested an influence on expression variability for some rare CNVs. Our straightforward CNV detection workflow can be easily applied to any NGS coverage data and helped to analyze CNVs in an ADME-NGS panel of 340 pharmacogenes to improve genotype-phenotype correlations.

MiR-155 and other microRNAs downregulate drug metabolizing cytochromes P450 in inflammation.

In conditions of acute and chronic inflammation hepatic detoxification capacity is severely impaired due to coordinated downregulation of drug metabolizing enzymes and transporters. Using global transcriptome analysis of liver tissue from donors with pathologically elevated C-reactive protein (CRP), we observed comparable extent of positive and negative acute phase response, where the top upregulated gene sets included immune response and defense pathways while downregulation occurred mostly in metabolic and catabolic pathways including many important drug metabolizing enzymes and transporters. We hypothesized that microRNAs (miRNA), which usually act as negative regulators of gene expression, contribute to this process. Microarray and quantitative real-time PCR analyses identified differentially expressed miRNAs in liver tissues from donors with elevated CRP, cholestasis, steatosis, or non-alcoholic steatohepatitis. Using luciferase reporter constructs harboring native and mutated 3'-untranslated gene regions, several predicted miRNA binding sites on RXRα (miR-130b-3p), CYP2C8 (miR-452-5p), CYP2C9 (miR-155-5p), CYP2C19 (miR-155-5p, miR-6807-5p), and CYP3A4 (miR-224-5p) were validated. HepaRG cells transfected with miRNA mimics showed coordinate reductions in mRNA levels and several cytochrome P450 enzyme activities particularly for miR-155-5p, miR-452-5p, and miR-6807-5p, the only miRNA that was deregulated in all four pathological conditions. Furthermore we observed strong negative correlations between liver tissue miRNA levels and hepatic CYP phenotypes. Since miR-155 is well known for its multifunctional roles in immunity, inflammation, and cancer, our data suggest that this and other miRNAs contribute to coordinated downregulation of drug metabolizing enzymes and transporters in inflammatory conditions.

Factors Affecting Interindividual Variability of Hepatic UGT2B17 Protein Expression Examined Using a Novel Specific Monoclonal Antibody.

Accurate quantification of the metabolic enzyme uridine diphospho-glucuronosyltransferase (UGT) UGT2B17 has been hampered by the high sequence identity with other UGT2B enzymes (as high as 94%) and by the lack of a specific antibody. Knowing the significance of the UGT2B17 pathway in drug and hormone metabolism and cancer, we developed a specific monoclonal antibody (EL-2B17mAb), initially validated by the lack of detection in liver microsomes of an individual carrying no UGT2B17 gene copy and in supersomes expressing UGT2B enzymes. Immunohistochemical detection in livers revealed strong labeling of bile ducts and variable labeling of hepatocytes. Expression levels assessed by immunoblotting were highly correlated to mass spectrometry-based quantification (r = 0.93), and three major expression patterns (absent, low, or high) were evidenced. Livers with very low expression were carriers of the functional rs59678213 G variant, located in the binding site for the transcription factor forkhead box A1 (FOXA1) of the UGT2B17 promoter. The highest level of expression was observed for individuals carrying at least one rs59678213 A allele. Multiple regression analysis indicated that the number of gene copies explained only 8% of UGT2B17 protein expression, 49% when adding rs59678213, reaching 54% when including sex. The novel EL-2B17mAb antibody allowed specific UGT2B17 quantification and exposed different patterns of hepatic expression. It further suggests that FOXA1 is a key driver of UGT2B17 expression in the liver. The availability of this molecular tool will help characterize the UGT2B17 level in various disease states and establish more precisely the contribution of the UGT2B17 enzyme to drug and hormone metabolism.

A New Panel-Based Next-Generation Sequencing Method for ADME Genes Reveals Novel Associations of Common and Rare Variants With Expression in a Human Liver Cohort.

We developed a panel-based NGS pipeline for comprehensive analysis of 340 genes involved in absorption, distribution, metabolism and excretion (ADME) of drugs, other xenobiotics, and endogenous substances. The 340 genes comprised phase I and II enzymes, drug transporters and regulator/modifier genes within their entire coding regions, adjacent intron regions and 5' and 3'UTR regions, resulting in a total panel size of 1,382 kbp. We applied the ADME NGS panel to sequence genomic DNA from 150 Caucasian liver donors with available comprehensive gene expression data. This revealed an average read-depth of 343 (range 27-811), while 99% of the 340 genes were covered on average at least 100-fold. Direct comparison of variant annotation with 363 available genotypes determined independently by other methods revealed an overall accuracy of >99%. Of 15,727 SNV and small INDEL variants, 12,022 had a minor allele frequency (MAF) below 2%, including 8,937 singletons. In total we found 7,273 novel variants. Functional predictions were computed for coding variants (n = 4,017) by three algorithms (Polyphen 2, Provean, and SIFT), resulting in 1,466 variants (36.5%) concordantly predicted to be damaging, while 1,019 variants (25.4%) were predicted to be tolerable. In agreement with other studies we found that less common variants were enriched for deleterious variants. Cis-eQTL analysis of variants with (MAF ≥ 2%) revealed significant associations for 90 variants in 31 genes after Bonferroni correction, most of which were located in non-coding regions. For less common variants (MAF < 2%), we applied the SKAT-O test and identified significant associations to gene expression for ADH1C and GSTO1. Moreover, our data allow comparison of functional predictions with additional phenotypic data to prioritize variants for further analysis.

The azole fungicide tebuconazole affects human CYP1A1 and CYP1A2 expression by an aryl hydrocarbon receptor-dependent pathway.

Tebuconazole, a member of the triazole group of fungicides, exerts hepatotoxicity in rodent studies. Knowledge on the molecular mechanisms underlying tebuconazole toxicity is limited. Previous studies suggest that activation of xenobiotic-sensing nuclear receptors plays a role in triazole fungicide-mediated hepatotoxicity. This study aimed to characterize the ability of tebuconazole to activate gene expression via the aryl hydrocarbon receptor (AHR). Results demonstrate a statistically significant induction of the AHR target genes CYP1A1 and CYP1A2 in HepG2 and HepaRG human liver cells in vitro at concentrations corresponding to tebuconazole tissue levels reached under subtoxic conditions in vivo. CYP1A1 and CYP1A2 induction was abolished in the presence of an AHR antagonist or in AHR-knockout HepaRG cells, substantiating the importance of the AHR for the observed effects. Although the results indicate that tebuconazole is a weak inducer of AHR-dependent genes, combined exposure of HepaRG cells to tebuconazole and the previously identified AHR agonist propiconazole showed additive effects on CYP1A1 and CYP1A2 expression. In summary, we demonstrate that AHR-downstream gene expression is affected by tebuconazole in an AHR-dependent manner. Data indicate that dose addition may be assumed for the assessment of AHR-related effects of triazole fungicide mixtures.

ß-Defensin 1 Is Prominent in the Liver and Induced During Cholestasis by Bilirubin and Bile Acids via Farnesoid X Receptor and Constitutive Androstane Receptor.

Background & aims: Knowledge about innate antimicrobial defense of the liver is limited. We investigated hepatic expression and regulation of antimicrobial peptides with focus on the human beta defensin-1 (hBD-1). Methods: Radial diffusion assay was used to analyze antimicrobial activity of liver tissue. Different defensins including hBD-1 and its activator thioredoxin-1 (TXN) were analyzed in healthy and cholestatic liver samples by qPCR and immunostaining. Regulation of hBD-1 expression was studied in vitro and in vivo using bile duct-ligated mice. Regulation of hBD-1 via bilirubin and bile acids (BAs) was studied using siRNA. Results: We found strong antimicrobial activity of liver tissue against Escherichia coli. As a potential mediator of this antimicrobial activity we detected high expression of hBD-1 and TXN in hepatocytes, whereas other defensins were minimally expressed. Using a specific antibody for the reduced, antimicrobially active form of hBD-1 we found hBD-1 in co-localization with TXN within hepatocytes. hBD-1 was upregulated in cholestasis in a graded fashion. In cholestatic mice hepatic AMP expression (Defb-1 and Hamp) was enhanced. Bilirubin and BAs were able to induce hBD-1 in hepatic cell cultures in vitro. Treatment with siRNA and/or agonists demonstrated that the farnesoid X receptor (FXR) mediates basal expression of hBD-1, whereas both constitutive androstane receptor (CAR) and FXR seem to be responsible for the induction of hBD-1 by bilirubin. Conclusion: hBD-1 is prominently expressed in hepatocytes. It is induced during cholestasis through bilirubin and BAs, mediated by CAR and especially FXR. Reduction by TXN activates hBD-1 to a potential key player in innate antimicrobial defense of the liver.

Epigenetics and MicroRNAs in Pharmacogenetics.

Germline pharmacogenetics has so far mainly studied common variants in "pharmacogenes," i.e., genes encoding drug metabolizing enzymes and transporters (DMET genes), certain auxiliary and regulatory genes, and drug target genes. Despite remarkable progress in understanding genetically determined differences in pharmacokinetics and pharmacodynamics of drugs, currently known common variants even in important pharmacogenes explain genetic variability only partially. This suggests "missing heritability" that may in part be due to rare variants in the classical pharmacogenes, but current evidence suggests that largely unexplored resources with potential for pharmacogenetics exist, both within already known pharmacogenes and in entirely new areas. In particular, recent studies suggest that epigenetic processes and noncoding RNAs, including mostly microRNAs (miRNAs), represent important and largely unexplored layers of DMET gene regulation that may fill some of the gaps in understanding interindividual variability and lead to new biomarkers. In this chapter we summarize recent advances in the understanding of genetic variability in epigenetic and miRNA-mediated processes with focus on their significance for DMET regulation and pharmacokinetic or pharmacological endpoints.

Unexpected Effects of Propiconazole, Tebuconazole, and Their Mixture on the Receptors CAR and PXR in Human Liver Cells.

Analyzing mixture toxicity requires an in-depth understanding of the mechanisms of action of its individual components. Substances with the same target organ, same toxic effect and same mode of action (MoA) are believed to cause additive effects, whereas substances with different MoAs are assumed to act independently. Here, we tested 2 triazole fungicides, propiconazole, and tebuconazole (Te), for individual and combined effects on liver toxicity-related endpoints. Both triazoles are proposed to belong to the same cumulative assessment group and are therefore thought to display similar and additive behavior. Our data show that Te is an antagonist of the constitutive androstane receptor (CAR) in rats and humans, while propiconazole is an agonist of this receptor. Both substances activate the pregnane X-receptor (PXR) and further induce mRNA expression of CYP3A4. CYP3A4 enzyme activity, however, is inhibited by propiconazole. For common targets of PXR and CAR, the activation of PXR by Te overrides CAR inhibition. In summary, propiconazole and Te affect different hepatotoxicity-relevant cellular targets and, depending on the individual endpoint analyzed, act via similar or dissimilar mechanisms. The use of molecular data based on research in human cell systems extends the picture to refine cumulative assessment group grouping and substantially contributes to the understanding of mixture effects of chemicals in biological systems.

Regulation of Drug Metabolism by the Interplay of Inflammatory Signaling, Steatosis, and Xeno-Sensing Receptors in HepaRG Cells.

Nonalcoholic fatty liver disease (NAFLD), which is characterized by triglyceride deposition in hepatocytes resulting from imbalanced lipid homeostasis, is of increasing concern in Western countries, along with progression to nonalcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis. Previous studies suggest a complex, mutual influence of hepatic fat accumulation, NASH-related inflammatory mediators, and drug-sensing receptors regulating xenobiotic metabolism. Here, we investigated the suitability of human HepaRG hepatocarcinoma cells as a model for NAFLD and NASH. Cells were incubated for up to 14 days with an oleate/palmitate mixture (125 µM each) and/or with 10 ng/ml of the inflammatory mediator interleukin-6 (IL-6). Effects of these conditions on the regulation of drug metabolism were studied using xenobiotic agonists of the aryl hydrocarbon receptor (AHR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), nuclear factor (erythroid-derived 2)-like 2, and peroxisome proliferator-activated receptor α (PPARα). Results underpin the suitability of HepaRG cells for NAFLD- and NASH-related research and constitute a broad-based analysis of the impact of hepatic fatty acid accumulation and inflammation on drug metabolism and its inducibility by xenobiotics. IL-6 exerted pronounced negative regulatory effects on basal as well as on PXR-, CAR-, and PPARα-, but not AHR-dependent induction of drug-metabolizing enzymes. This inhibition was related to diminished transactivation potential of the respective receptors rather than to reduced transcription of nuclear receptor-encoding mRNAs. The most striking effects of IL-6 and/or fatty acid treatment were observed in HepaRG cells after 14 days of treatment, making these cultures appear a suitable model for studying the relationship of fatty acid accumulation, inflammation, and xenobiotic-induced drug metabolism.

Direct Quantification of Cytochromes P450 and Drug Transporters-A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates.

The quantification of drug metabolizing enzymes and transporters has recently been revolutionized on the basis of targeted proteomic approaches. Isotope-labeled peptides are used as standards for the quantification of the corresponding proteins in enzymatically fragmented samples. However, hurdles in these approaches are low throughput and tedious sample prefractionation steps prior to mass spectrometry (MS) readout. We have developed an assay platform using sensitive and selective immunoprecipitation coupled with mass spectrometric readout allowing the quantification of proteins directly from whole cell lysates using less than 20,000 cells per analysis. Peptide group-specific antibodies (triple X proteomics antibodies) enable the enrichment of proteotypic peptides sharing a common terminus. These antibodies were employed to establish a MS-based immunoassay panel for the quantification of 14 cytochrome P450 (P450) enzymes and nine transporters. We analyzed the P450 enzyme and transporter levels in genotyped liver tissue homogenates and microsomes, and in samples from a time course induction experiment in human hepatocytes addressing different induction pathways. For the analysis of P450 enzymes and transporters only a minute amount of sample is required and no prefractionation is necessary, thus the assay platform bears the potential to bridge cell culture model experiments and results from whole organ tissue studies.

Systemic regulation of bilirubin homeostasis: Potential benefits of hyperbilirubinemia.

Neurotoxic bilirubin is the end product of heme catabolism in mammals. Bilirubin is solely conjugated by uridine diphospho-glucuronosyltransferase 1A1, which is a membrane-bound enzyme that catalyzes the transfer of glucuronic acid. Due to low function of hepatic and intestinal uridine diphospho-glucuronosyltransferase 1A1 during the neonatal period, human neonates develop mild to severe physiological hyperbilirubinemia. Accumulation of bilirubin in the brain leads to the onset of irreversible brain damage, termed kernicterus. Breastfeeding is one of the most significant factors that increase the risk of developing kernicterus in infants. Why does this most natural way of feeding increase the risk of brain damage or even death? This question leads to the hypothesis that breast milk-induced hyperbilirubinemia might bring certain benefits that outweigh those risks. While bilirubin is neurotoxic and cytotoxic, this compound is also a potent antioxidant. There are studies showing improved clinical conditions in patients with hyperbilirubinemia. Accumulating evidence also shows that genetic polymorphisms linked to hyperbilirubinemia are beneficial against various diseases. In this review article, we first introduce the production, metabolism, and transport of bilirubin. We then discuss the potential benefits of neonatal and adult hyperbilirubinemia. Finally, epigenetic factors as well as metabolomic information associated with hyperbilirubinemia are described. This review article advances the understanding of the physiological importance of the paradoxical compound bilirubin. (Hepatology 2018;67:1609-1619).

The formation of estrogen-like tamoxifen metabolites and their influence on enzyme activity and gene expression of ADME genes.

Tamoxifen, a standard therapy for breast cancer, is metabolized to compounds with anti-estrogenic as well as estrogen-like action at the estrogen receptor. Little is known about the formation of estrogen-like metabolites and their biological impact. Thus, we characterized the estrogen-like metabolites tamoxifen bisphenol and metabolite E for their metabolic pathway and their influence on cytochrome P450 activity and ADME gene expression. The formation of tamoxifen bisphenol and metabolite E was studied in human liver microsomes and Supersomes™. Cellular metabolism and impact on CYP enzymes was analyzed in upcyte® hepatocytes. The influence of 5 µM of tamoxifen, anti-estrogenic and estrogen-like metabolites on CYP activity was measured by HPLC MS/MS and on ADME gene expression using RT-PCR analyses. Metabolite E was formed from tamoxifen by CYP2C19, 3A and 1A2 and from desmethyltamoxifen by CYP2D6, 1A2 and 3A. Tamoxifen bisphenol was mainly formed from (E)- and (Z)-metabolite E by CYP2B6 and CYP2C19, respectively. Regarding phase II metabolism, UGT2B7, 1A8 and 1A3 showed highest activity in glucuronidation of tamoxifen bisphenol and metabolite E. Anti-estrogenic metabolites (Z)-4-hydroxytamoxifen, (Z)-endoxifen and (Z)-norendoxifen inhibited the activity of CYP2C enzymes while tamoxifen bisphenol consistently induced CYPs similar to rifampicin and phenobarbital. On the transcript level, highest induction up to 5.6-fold was observed for CYP3A4 by tamoxifen, (Z)-4-hydroxytamoxifen, tamoxifen bisphenol and (E)-metabolite E. Estrogen-like tamoxifen metabolites are formed in CYP-dependent reactions and are further metabolized by glucuronidation. The induction of CYP activity by tamoxifen bisphenol and the inhibition of CYP2C enzymes by anti-estrogenic metabolites may lead to drug-drug-interactions.