Synergistic and antagonistic interactions of binary mixtures of polycyclic aromatic hydrocarbons in the upregulation of CYP1 activity and mRNA levels in precision-cut rat liver slices.

The current studies investigate whether synergistic or antagonistic interactions in the upregulation of CYP1 activity occur in binary mixtures of polycyclic aromatic hydrocarbons (PAHs) involving benzo[a]pyrene and five other structurally diverse PAHs of varying carcinogenic activity. Precision-cut rat liver slices were incubated with benzo[a]pyrene alone or in combination with a range of concentrations of a second PAH, and ethoxyresorufin O-deethylase, CYP1A1 and CYP1B1 mRNA levels determined. Concurrent incubation of benzo[a]pyrene with either dibenzo[a,h]anthracene or fluoranthene in liver slices led to a synergistic interaction, at least at low concentrations, in that ethoxyresorufin O-deethylase activity was statistically higher than the added effects when the slices were incubated with the individual compounds. In contrast, benzo[b]fluoranthene and, at high doses only, dibenzo[a,l]pyrene gave rise to antagonism, whereas 1-methylphenanthrene had no effect at all concentrations studied. When CYP1A1 mRNA levels were monitored, benzo[b]fluoranthene gave rise to an antagonistic response when incubated with benzo[a]pyrene, whereas all other compounds displayed synergism, with 1-methylphenathrene being the least effective. A similar picture emerged when CYP1B1 mRNA levels were determined, though the effects were less pronounced. In conclusion, it has been demonstrated that the benzo[a]pyrene-mediated upregulation of CYP1, at the mRNA and activity levels, is synergistically and antagonistically modulated by other PAHs. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 764-775, 2017.

Fatty acid binding proteins: tissue-specific functions in health and disease.

PURPOSE OF REVIEW: The purpose of this study is to review recent evidence for the role of the cytosolic fatty acid binding proteins (FABPs) as central regulators of whole-body metabolic control. RECENT FINDINGS: Dysregulated FABPs have been associated with a number of diseases, including obesity and nonalcoholic fatty liver disease (FABP1, FABP2, FABP4), cardiovascular risk (FABP3) and cancer (FABP5, FABP7). As underlying mechanisms become better understood, FABPs may represent novel biomarkers for therapeutic targets. In addition, the role of FABPs as important signalling molecules has also been highlighted in recent years; for example, FABP3 may act as a myokine, matching whole-body metabolism to muscular energy demands and FABP4 functions as an adipokine in regulating macrophage and adipocyte interactions during inflammation. SUMMARY: In addition to their traditional role as fatty acid trafficking proteins, increasing evidence supports the role of FABPs as important controllers of global metabolism, with their dysregulation being linked to a host of metabolic diseases.

Site-specific, covalent incorporation of Tus, a DNA-binding protein, on ionic-complementary self-assembling peptide hydrogels using transpeptidase Sortase A as a conjugation tool†Dedicated to the memory of Joachim H. G. Steinke.‡Electronic supplementary information (ESI) available: Further experimental data. See DOI: 10.1039/c3sm00131hClick here for additional data file.

The site-specific conjugation of DNA-binding protein (Tus) to self-assembling peptide FEFEFKFKK was demonstrated. Rheology studies and TEM of the corresponding hydrogels (including PNIPAAm-containing systems) showed no significant variation in properties and hydrogel morphology compared to FEFEFKFKK. Critically, we demonstrate that Tus is accessible within the gel network displaying DNA-binding properties.

Linking physiologically-based pharmacokinetic and genome-scale metabolic networks to understand estradiol biology.

BACKGROUND: Estrogen is a vital hormone that regulates many biological functions within the body. These include roles in the development of the secondary sexual organs in both sexes, plus uterine angiogenesis and proliferation during the menstrual cycle and pregnancy in women. The varied biological roles of estrogens in human health also make them a therapeutic target for contraception, mitigation of the adverse effects of the menopause, and treatment of estrogen-responsive tumours. In addition, endogenous (e.g. genetic variation) and external (e.g. exposure to estrogen-like chemicals) factors are known to impact estrogen biology. To understand how these multiple factors interact to determine an individual's response to therapy is complex, and may be best approached through a systems approach. METHODS: We present a physiologically-based pharmacokinetic model (PBPK) of estradiol, and validate it against plasma kinetics in humans following intravenous and oral exposure. We extend this model by replacing the intrinsic clearance term with: a detailed kinetic model of estrogen metabolism in the liver; or, a genome-scale model of liver metabolism. Both models were validated by their ability to reproduce clinical data on estradiol exposure. We hypothesise that the enhanced mechanistic information contained within these models will lead to more robust predictions of the biological phenotype that emerges from the complex interactions between estrogens and the body. RESULTS: To demonstrate the utility of these models we examine the known drug-drug interactions between phenytoin and oral estradiol. We are able to reproduce the approximate 50% reduction in area under the concentration-time curve for estradiol associated with this interaction. Importantly, the inclusion of a genome-scale metabolic model allows the prediction of this interaction without directly specifying it within the model. In addition, we predict that PXR activation by drugs results in an enhanced ability of the liver to excrete glucose. This has important implications for the relationship between drug treatment and metabolic syndrome. CONCLUSIONS: We demonstrate how the novel coupling of PBPK models with genome-scale metabolic networks has the potential to aid prediction of drug action, including both drug-drug interactions and changes to the metabolic landscape that may predispose an individual to disease development.

Integration of Genome Scale Metabolic Networks and Gene Regulation of Metabolic Enzymes With Physiologically Based Pharmacokinetics.

The scope of physiologically based pharmacokinetic (PBPK) modeling can be expanded by assimilation of the mechanistic models of intracellular processes from systems biology field. The genome scale metabolic networks (GSMNs) represent a whole set of metabolic enzymes expressed in human tissues. Dynamic models of the gene regulation of key drug metabolism enzymes are available. Here, we introduce GSMNs and review ongoing work on integration of PBPK, GSMNs, and metabolic gene regulation. We demonstrate example models.

Generation of 2,000 breast cancer metabolic landscapes reveals a poor prognosis group with active serotonin production.

A major roadblock in the effective treatment of cancers is their heterogeneity, whereby multiple molecular landscapes are classified as a single disease. To explore the contribution of cellular metabolism to cancer heterogeneity, we analyse the Metabric dataset, a landmark genomic and transcriptomic study of 2,000 individual breast tumours, in the context of the human genome-scale metabolic network. We create personalized metabolic landscapes for each tumour by exploring sets of active reactions that satisfy constraints derived from human biochemistry and maximize congruency with the Metabric transcriptome data. Classification of the personalized landscapes derived from 997 tumour samples within the Metabric discovery dataset reveals a novel poor prognosis cluster, reproducible in the 995-sample validation dataset. We experimentally follow mechanistic hypotheses resulting from the computational study and establish that active serotonin production is a major metabolic feature of the poor prognosis group. These data support the reconsideration of concomitant serotonin-specific uptake inhibitors treatment during breast cancer chemotherapy.

Activation of the Farnesoid X-receptor in breast cancer cell lines results in cytotoxicity but not increased migration potential.

Breast cancer is the commonest form of cancer in women, but successful treatment is confounded by the heterogeneous nature of breast tumours: Effective treatments exist for hormone-sensitive tumours, but triple-negative breast cancer results in poor survival. An area of increasing interest is metabolic reprogramming, whereby drug-induced alterations in the metabolic landscape of a tumour slow tumour growth and/or increase sensitivity to existing therapeutics. Nuclear receptors are transcription factors central to the expression of metabolic and transport proteins, and thus represent potential targets for metabolic reprogramming. We show that activation of the nuclear receptor FXR, either by its endogenous ligand CDCA or the synthetic GW4064, leads to cell death in four breast cancer cell lines with distinct phenotypes: MCF-10A (normal), MCF-7 (receptor positive), MDA-MB-231 and MDA-MB-468 (triple negative). Furthermore, we show that the mechanism of cell death is predominantly through the intrinsic apoptotic pathway. Finally, we demonstrate that FXR agonists do not stimulate migration in breast cancer cell lines, an important potential adverse effect. Together, our data support the continued examination of FXR agonists as a novel class of therapeutics for the treatment of breast cancer.

Inhibition of prenyltransferase activity by statins in both liver and muscle cell lines is not causative of cytotoxicity.

As inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, statins are an important first-line treatment for hypercholesterolemia. However, a recognized side-effect of statin therapy is myopathy, which in severe cases can present as potentially fatal rhabdomyolysis. This represents an important impediment to successful statin therapy, and despite decades of research the molecular mechanisms underlying this side-effect remain unclear. Current evidence supports a role for reduced levels of mevalonate pathway intermediates, with the most accepted hypothesis being a reduction in isoprenoids formation, leading to faulty post-translational modifications of membrane-associated proteins. We have undertaken a comprehensive analysis of the impact of nine statins on two human cell lines; Huh7 hepatoma and RD rhabdomyosarcoma. In both cell lines, concentration-dependent inhibition of prenylation was observed for cerivastatin and simvastatin, which could be rescued with the pathway intermediate mevalonate; in general, muscle cells were more sensitive to this effect, as measured by the levels of unprenylated Rap1A, a marker for prenylation by geranylgeranyl transferase I. Concentration-dependent toxicity was observed in both cell lines, with muscle cells again being more sensitive. Importantly, there was no correlation between inhibition of prenylation and cell toxicity, suggesting they are not causally linked. The lack of a causal relationship was confirmed by the absence of cytotoxicity in all cell lines following exposure to specific inhibitors of geranylgeranyl transferases I and II, and farnesyl transferase. As such, we provide strong evidence against the commonly accepted hypothesis linking inhibition of prenylation and statin-mediated toxicity, with the two processes likely to be simultaneous but independent.

Evaluation of the toxicological relevance of CYP3A4 induction.

CYP3A4 is the most abundant cytochrome P450 in human liver, comprising approximately 30% of the total liver P450 content. This enzyme has an important role in endogenous processes, most notably steroid catabolism, and also plays a fundamental role in the metabolism of more than half of the clinically used drugs currently prescribed. The majority of CYP3A substrates are also capable of upregulating CYP3A activity, mainly through transcriptional activation. The molecular mechanisms that underlie the transcriptional activation of CYP3A4 are complex, with many steroid hormone nuclear receptors, including GR, PXR, VDR and CAR, playing a role in these mechanisms. However, the net result of transcriptional activation is an increase in the metabolism of the inducing compounds and, therefore, increased clearance. An important side effect of this transcriptional activation is that co-administered chemicals metabolized by CYP3A may also have their pharmacokinetics altered. Such changes can result in reduced clinical efficacy of drugs, resulting in poor patient response, or the development of an adverse drug response. This review will examine examples of established interactions caused through transcriptional activation of CYP3A4, and speculate on whether such effects are clinically important and should be considered during the design of treatment regimes or, alternatively, are relatively minor and cause little physiological effects.

Gene expression changes in rat liver following exposure to liver growth agents: role of Kupffer cells in xenobiotic-mediated liver growth.

Many xenobiotics are known to cause liver enlargement and hepatocarcinogenesis in rats, although the molecular mechanisms that underlie this effect remain largely undefined. Human exposure to several of these compounds, including glucocorticoids and peroxisome proliferators may be significant, due to their use in both pharmaceutical and industrial processes. It is therefore important to elucidate the molecular mechanisms underlying this abnormal liver enlargement in rats, as this will enable more accurate extrapolation of the possible outcomes of human exposure. Male Sprague-Dawley rats were dosed with the peroxisome proliferator Wy-14,643 and changes in liver gene expression examined using subtractive suppression hybridisation examined either 12 of 24hr later. Twenty-five transcripts were identified which showed differential gene expression in liver following exposure to Wy-14,643. Biochemical indices of liver growth (DNA synthesis, apoptosis) showed that these changes correlated with the initiation of liver enlargement. Rats were next treated with either Wy-14,643, cyproterone acetate and dexamethasone, chemically and mechanistically-distinct hepatomegalic compounds. Carboxylesterase and Kupffer cell receptor mRNA levels were seen to alter in a qualitatively similar fashion for all three compounds, and in a liver specific fashion. In addition, these changes correlated with a decrease in the density of Kupffer cells within the liver, which are known to release mitogenic cytokines, and have been linked to Wy-14,643-induced cell proliferation. We therefore propose that Kupffer cells play a role in a general mechanism of xenobiotic-mediated liver enlargement.

Receptor-dependent regulation of the CYP3A4 gene.

A CYP3A4 promoter-reporter gene construct has been used to assess the ability of 16 known (in vivo) and putative (in vitro) inducers to transactivate a CYP3A4 reporter gene in HepG2 cells. With the exception of pravastatin, the remaining 15 compounds transactivated the CYP3A4 reporter gene with differing inductive abilities (I(max):EC(50)) over two orders of magnitude, ranging from 1.1 (phenytoin) to 222.9 (lovastatin) in a receptor-supplemented system and it is proposed that the lack of response to pravastatin is due to loss of the known hepatic uptake transporter in HepG2 cells. In addition, reporter gene assays were used to investigate two promoter mutants namely a T to C change at -191 bp in the hepatic nuclear factor 3 binding site (HNF-3, -187 to -194 bp) and an A to G change at -205 bp in the oestrogen response element (ERE, -202 to -212 bp), which conferred differential responsiveness to steroid and xenobiotic inducers.

Systems biology approaches for studying the pathogenesis of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a progressive disease of increasing public health concern. In western populations the disease has an estimated prevalence of 20%-40%, rising to 70%-90% in obese and type II diabetic individuals. Simplistically, NAFLD is the macroscopic accumulation of lipid in the liver, and is viewed as the hepatic manifestation of the metabolic syndrome. However, the molecular mechanisms mediating both the initial development of steatosis and its progression through non-alcoholic steatohepatitis to debilitating and potentially fatal fibrosis and cirrhosis are only partially understood. Despite increased research in this field, the development of non-invasive clinical diagnostic tools and the discovery of novel therapeutic targets has been frustratingly slow. We note that, to date, NAFLD research has been dominated by in vivo experiments in animal models and human clinical studies. Systems biology tools and novel computational simulation techniques allow the study of large-scale metabolic networks and the impact of their dysregulation on health. Here we review current systems biology tools and discuss the benefits to their application to the study of NAFLD. We propose that a systems approach utilising novel in silico modelling and simulation techniques is key to a more comprehensive, better targeted NAFLD research strategy. Such an approach will accelerate the progress of research and vital translation into clinic.

Primary liver cells cultured on carbon nanotube substrates for liver tissue engineering and drug discovery applications.

Here, we explore the use of two- and three-dimensional scaffolds of multiwalled-carbon nanotubes (MWNTs) for hepatocyte cell culture. Our objective is to study the use of these scaffolds in liver tissue engineering and drug discovery. In our experiments, primary rat hepatocytes, the parenchymal (main functional) cell type in the liver, were cultured on aligned nanogrooved MWNT sheets, MWNT yarns, or standard 2-dimensional culture conditions as a control. We find comparable cell viability between all three culture conditions but enhanced production of the hepatocyte-specific marker albumin for cells cultured on MWNTs. The basal activity of two clinically relevant cytochrome P450 enzymes, CYP1A2 and CYP3A4, are similar on all substrates, but we find enhanced induction of CYP1A2 for cells on the MWNT sheets. Our data thus supports the use of these substrates for applications including tissue engineering and enhancing liver-specific functions, as well as in in vitro model systems with enhanced predictive capability in drug discovery and development.

Conserved valproic-acid-induced lipid droplet formation in Dictyostelium and human hepatocytes identifies structurally active compounds.

Lipid droplet formation and subsequent steatosis (the abnormal retention of lipids within a cell) has been reported to contribute to hepatotoxicity and is an adverse effect of many pharmacological agents including the antiepileptic drug valproic acid (VPA). In this study, we have developed a simple model system (Dictyostelium discoideum) to investigate the effects of VPA and related compounds in lipid droplet formation. In mammalian hepatocytes, VPA increases lipid droplet accumulation over a 24-hour period, giving rise to liver cell damage, and we show a similar effect in Dictyostelium following 30 minutes of VPA treatment. Using (3)H-labelled polyunsaturated (arachidonic) or saturated (palmitic) fatty acids, we shown that VPA treatment of Dictyostelium gives rise to an increased accumulation of both types of fatty acids in phosphatidylcholine, phosphatidylethanolamine and non-polar lipids in this time period, with a similar trend observed in human hepatocytes (Huh7 cells) labelled with [(3)H]arachidonic acid. In addition, pharmacological inhibition of ß-oxidation in Dictyostelium phenocopies fatty acid accumulation, in agreement with data reported in mammalian systems. Using Dictyostelium, we then screened a range of VPA-related compounds to identify those with high and low lipid-accumulation potential, and validated these activities for effects on lipid droplet formation by using human hepatocytes. Structure-activity relationships for these VPA-related compounds suggest that lipid accumulation is independent of VPA-catalysed teratogenicity and inositol depletion. These results suggest that Dictyostelium could provide both a novel model system for the analysis of lipid droplet formation in human hepatocytes and a rapid method for identifying VPA-related compounds that show liver toxicology.

Transcriptomic and phylogenetic analysis of Kpna genes: a family of nuclear import factors modulated in xenobiotic-mediated liver growth.

OBJECTIVES: We have identified a member of the karyopherin (importin) alpha family of nuclear import factors as being modulated in rat liver following exposure to the hypolipidaemic and liver growth agent Wy-14,643. To examine the hypothetical role of this protein family as a checkpoint in receptor-mediated signalling, we characterized the rat karyopherin alpha (Kpna) gene family and present cDNA sequences and gene structures for all six rat Kpna genes. Further, we have assembled a comprehensive panel of Kpna coding regions from a range of metazoa, which we have subjected to phylogenetic analysis: This represents by far the most complete phylogenetic study of metazoan karyopherins, including several evolutionary intermediates not previously examined. The phylogeny reveals three Kpna subfamilies with distinct, conserved gene structures, shedding light on the evolutionary origins of this multigene family in metazoa. METHODS AND RESULTS: Using quantitative PCR, we have analysed Kpna transcript levels in 44 rat tissues; Kpna transcripts show a wide variation in their distribution both in absolute and relative terms, suggestive of specialized roles for each member. We also demonstrate that Kpna genes are regulated in rat liver and isolated hepatocytes in a xenobiotic-specific manner for a number of chemically distinct liver growth agents. CONCLUSIONS: In light of the crucial role of nuclear import in mediating the genomic changes elicited through nuclear receptor activation, we postulate that changes in the levels of specific karyopherins alpha during xenobiotic-mediated liver growth represent an important component of the cellular response to the external stimuli that trigger these events.

Impact of transcription factor profile and chromatin conformation on human hepatocyte CYP3A gene expression.

Recent data have made it increasingly clear that the gene expression profile of a cell system, and its alteration in response to external stimuli, is highly dependent on both the higher order chromatin structure of the genome and the interaction of gene products in interpreting stimuli. To further explore this phenomenon, we have examined the role of both of these factors in controlling xenobiotic-mediated gene expression changes in primary and transformed human hepatocytes (HuH7). Using quantitative polymerase chain reaction, expression levels of several transcription factors implicated in the liver-specific regulation of the CYP3A gene family were examined in human adult and fetal liver RNA samples. These expression profiles were then compared with those obtained from both primary and transformed human hepatocytes, showing that, in general, cultured cells exhibit a distinct profile compared with either the fetal or adult samples. Transcriptome profiles before and after exposure to the CYP3A transcriptional activators rifampicin, dexamethasone, pregnane-16alpha-carbonitrile, and phenobarbital were subsequently examined. Whereas exposure to these compounds elicited a dose-dependent increase in CYP3A transcription in primary hepatocytes, no alteration in expression levels was observed for the hepatoma cell line HuH7. Alteration in the expression levels of pregnane X receptor and chicken ovalbumin upstream promoter transcription factor I, and the disruption of higher order chromatin within HuH7 cells altered CYP3A expression and/or activation by xenobiotics toward that observed in primary hepatocytes. These data provide potential roles for these two processes in regulating CYP3A expression in vivo.