Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing.

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

Hepatocyte Nuclear Factor 4α (HNF4α) Plays a Controlling Role in Expression of the Retinoic Acid Receptor ß (RARß) Gene in Hepatocytes.

HNF4α, a member of the nuclear receptor superfamily, regulates the genes involved in lipid and glucose metabolism. The expression of the RARß gene in the liver of HNF4α knock-out mice was higher versus wildtype controls, whereas oppositely, RARß promoter activity was 50% reduced by the overexpression of HNF4α in HepG2 cells, and treatment with retinoic acid (RA), a major metabolite of vitamin A, increased RARß promoter activity 15-fold. The human RARß2 promoter contains two DR5 and one DR8 binding motifs, as RA response elements (RARE) proximal to the transcription start site. While DR5 RARE1 was previously reported to be responsive to RARs but not to other nuclear receptors, we show here that mutation in DR5 RARE2 suppresses the promoter response to HNF4α and RARα/RXRα. Mutational analysis of ligand-binding pocket amino acids shown to be critical for fatty acid (FA) binding indicated that RA may interfere with interactions of FA carboxylic acid headgroups with side chains of S190 and R235, and the aliphatic group with I355. These results could explain the partial suppression of HNF4α transcriptional activation toward gene promoters that lack RARE, including APOC3 and CYP2C9, while conversely, HNF4α may bind to RARE sequences in the promoter of the genes such as CYP26A1 and RARß, activating these genes in the presence of RA. Thus, RA could act as either an antagonist towards HNF4α in genes lacking RAREs, or as an agonist for RARE-containing genes. Overall, RA may interfere with the function of HNF4α and deregulate HNF4α targets genes, including the genes important for lipid and glucose metabolism.

An FDA/CDER perspective on nonclinical testing strategies: Classical toxicology approaches and new approach methodologies (NAMs).

Nonclinical testing of human pharmaceuticals is conducted to assess the safety of compounds to be studied in human clinical trials and for marketing of new drugs. Although there is no exact number and type of nonclinical studies required for safety assessments, as there is inherent flexibility for each new compound, the traditional approach is outlined in various FDA and ICH guidance documents and involves a combination of in vitro assays and whole animal testing methods. Recent advances in science have led to the emergence of numerous new approach methodologies (NAMs) for nonclinical testing that are currently being used in various aspects of drug development. Traditional nonclinical testing methods can predict clinical outcomes, although improvements in these methods that can increase predictivity of clinical outcomes are encouraged and needed. This paper discusses FDA/CDER's view on the opportunities and challenges of using NAMs in drug development especially for regulatory purposes, and also includes examples where NAMs are currently being used in nonclinical safety assessments and where they may supplement and/or enhance current testing methods. FDA/CDER also encourages communication with stakeholders regarding NAMs and is committed to exploring the use of NAMs to improve regulatory efficiency and potentially expedite drug development.

Hepatocyte nuclear factor 4α regulates the expression of intestinal epithelial Na+/H+ exchanger isoform 3.

Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-α, IFN-γ, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF-4α) binding site that is evolutionarily conserved in several species suggesting that HNF-4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4α are not known. We investigated the regulation of NHE3 gene expression by HNF-4α in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4α-null ( Hnf4αΔIEpC) and control ( Hnf4αfl/fl) mice. HNF-4α knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4α directly interacts with the HNF-4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4α motif decreased, whereas ectopic overexpression of HNF-4α increased, NHE3 promoter activity. Furthermore, loss of HNF-4α in Hnf4αΔIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4α directly regulates NHE3 promoter activity and its basal expression in the intestine.

Hepatocyte-specific PPARA expression exclusively promotes agonist-induced cell proliferation without influence from nonparenchymal cells.

Peroxisome proliferator-activated receptor-α (PPARA) is a nuclear transcription factor and key mediator of systemic lipid metabolism. Prolonged activation in rodents causes hepatocyte proliferation and hepatocellular carcinoma. Little is known about the contribution of nonparenchymal cells (NPCs) to PPARA-mediated cell proliferation. NPC contribution to PPARA agonist-induced hepatomegaly was assessed in hepatocyte (Ppara△Hep)- and macrophage (Ppara△Mac)-specific Ppara null mice. Mice were treated with the agonist Wy-14643 for 14 days, and response of conditional null mice was compared with conventional knockout mice (Ppara-/- ). Wy-14643 treatment caused weight loss and severe hepatomegaly in wild-type and Ppara△Mac mice, and histological analysis revealed characteristic hepatocyte swelling; Ppara△Hep and Ppara-/- mice were protected from these effects. Ppara△Mac serum chemistries, as well as aspartate aminotransferase and alanine aminotransferase levels, matched wild-type mice. Agonist-treated Ppara△Hep mice had elevated serum cholesterol, phospholipids, and triglycerides when compared with Ppara-/- mice, indicating a possible role for extrahepatic PPARA in regulating circulating lipid levels. BrdU labeling confirmed increased cell proliferation only in wild-type and Ppara△Mac mice. Macrophage PPARA disruption did not impact agonist-induced upregulation of lipid metabolism, cell proliferation, or DNA damage and repair-related gene expression, whereas gene expression was repressed in Ppara△Hep mice. Interestingly, downregulation of inflammatory cytokines IL-15 and IL-18 was dependent on macrophage PPARA. Cell type-specific regulation of target genes was confirmed in primary hepatocytes and Kupffer cells. These studies conclusively show that cell proliferation is mediated exclusively by PPARA activation in hepatocytes and that Kupffer cell PPARA has an important role in mediating the anti-inflammatory effects of PPARA agonists.

Hepatic sirtuin 1 is dispensable for fibrate-induced peroxisome proliferator-activated receptor-α function in vivo.

Peroxisome proliferator-activated receptor-α (PPARα) mediates metabolic remodeling, resulting in enhanced mitochondrial and peroxisomal ß-oxidation of fatty acids. In addition to the physiological stimuli of fasting and high-fat diet, PPARα is activated by the fibrate class of drugs for the treatment of dyslipidemia. Sirtuin 1 (SIRT1), an important regulator of energy homeostasis, was downregulated in fibrate-treated wild-type mice, suggesting PPARα regulation of Sirt1 gene expression. The impact of SIRT1 loss on PPARα functionality in vivo was assessed in hepatocyte-specific knockout mice that lack the deacetylase domain of SIRT1 (Sirt1(ΔLiv)). Knockout mice were treated with fibrates or fasted for 24 h to activate PPARα. Basal expression of the PPARα target genes Cyp4a10 and Cyp4a14 was reduced in Sirt1(ΔLiv) mice compared with wild-type mice. However, no difference was observed between wild-type and Sirt1(ΔLiv) mice in either fasting- or fibrate-mediated induction of PPARα target genes. Similar to the initial results, there was no difference in fibrate-activated PPARα gene induction. To assess the relationship between SIRT1 and PPARα in a pathophysiological setting, Sirt1(ΔLiv) mice were maintained on a high-fat diet for 14 wk, followed by fibrate treatment. Sirt1(ΔLiv) mice exhibited increased body mass compared with control mice. In the context of a high-fat diet, Sirt1(ΔLiv) mice did not respond to the cholesterol-lowering effects of the fibrate treatment. However, there were no significant differences in PPARα target gene expression. These results suggest that, in vivo, SIRT1 deacetylase activity does not significantly impact induced PPARα activity.

Metabolomics reveals aging-associated attenuation of noninvasive radiation biomarkers in mice: potential role of polyamine catabolism and incoherent DNA damage-repair.

Development of methods for rapid screening and stratification of subjects after exposure is an integral part of countermeasures against radiation. The potential demographic and exposure history-related heterogeneity of exposed populations warrants robust biomarkers that withstand and reflect such differences. In this study, the effect of aging and repeated exposure on the metabolic response to sublethal irradiation was examined in mice using UPLC-ESI-QTOF mass spectrometry. Aging attenuated postexposure elevation in excretions of DNA damage biomarkers as well as N(1)-acetylspermidine. Although N(1)-acetylspermidine and 2'-deoxyuridine elevation was highly correlated in all age groups, xanthine and N(1)-acetylspermidine elevation was poorly correlated in older mice. These results may reflect the established decline in DNA damage-repair efficiency associated with aging and indicate a novel role for polyamine metabolism in the process. Although repeated irradiation at long intervals did not affect the elevation of N(1)-acetylspermidine, 2'-deoxyuridine, and xanthine, it did significantly attenuate the elevation of 2'-deoxycytidine and thymidine compared to a single exposure. However, these biomarkers were found to identify exposed subjects with accuracy ranging from 82% (xanthosine) to 98% (2'-deoxyuridine), irrespective of their age and exposure history. This indicates that metabolic biomarkers can act as robust noninvasive signatures of sublethal radiation exposure.

Suppression of hepatocyte proliferation by hepatocyte nuclear factor 4α in adult mice.

Hepatocyte nuclear factor 4α (HNF4α) regulates genes involved in lipid and bile acid synthesis, gluconeogenesis, amino acid metabolism, and blood coagulation. In addition to its metabolic role, HNF4α is critical for hepatocyte differentiation, and loss of HNF4α is associated with hepatocellular carcinoma. The hepatocyte-specific Hnf4a knock-out mouse develops severe hepatomegaly and steatosis resulting in premature death, thereby limiting studies of the role of this transcription factor in the adult animal. In addition, gene compensation may complicate analysis of the phenotype of these mice. To overcome these issues, an acute Hnf4a knock-out mouse model was generated through use of the tamoxifen-inducible ErT2cre coupled to the serum albumin gene promoter. Microarray expression analysis revealed up-regulation of genes associated with proliferation and cell cycle control only in the acute liver-specific Hnf4α-null mouse. BrdU and ki67 staining confirmed extensive hepatocyte proliferation in this model. Proliferation was associated with induction of the hepatomitogen Bmp7 as well as reduced basal apoptotic activity. The p53/p63 apoptosis effector gene Perp was further identified as a direct HNF4α target gene. These data suggest that HNF4α maintains hepatocyte differentiation in the adult healthy liver, and its loss may directly contribute to hepatocellular carcinoma development, thus indicating this factor as a possible liver tumor suppressor gene.

Cytochrome P450 regulation by α-tocopherol in Pxr-null and PXR-humanized mice.

The pregnane X receptor (PXR) has been postulated to play a role in the metabolism of α-tocopherol owing to the up-regulation of hepatic cytochrome P450 (P450) 3A in human cell lines and murine models after α-tocopherol treatment. However, in vivo studies confirming the role of PXR in α-tocopherol metabolism in humans presents significant difficulties and has not been performed. PXR-humanized (hPXR), wild-type, and Pxr-null mouse models were used to determine whether α-tocopherol metabolism is influenced by species-specific differences in PXR function in vivo. No significant difference in the concentration of the major α-tocopherol metabolites was observed among the hPXR, wild-type, and Pxr-null mice through mass spectrometry-based metabolomics. Gene expression analysis revealed significantly increased expression of Cyp3a11 as well as several other P450s only in wild-type mice, suggesting species-specificity for α-tocopherol activation of PXR. Luciferase reporter assay confirmed activation of mouse PXR by α-tocopherol. Analysis of the Cyp2c family of genes revealed increased expression of Cyp2c29, Cyp2c37, and Cyp2c55 in wild-type, hPXR, and Pxr-null mice, which suggests PXR-independent induction of Cyp2c gene expression. This study revealed that α-tocopherol is a partial agonist of PXR and that PXR is necessary for Cyp3a induction by α-tocopherol. The implications of a novel role for α-tocopherol in Cyp2c gene regulation are also discussed.

Disruption of thioredoxin reductase 1 protects mice from acute acetaminophen-induced hepatotoxicity through enhanced NRF2 activity.

The critical importance of glutathione in mitigating the deleterious effects of electrophile generating drugs such as acetaminophen (APAP) is well established. However, the role of other antioxidant systems, such as that provided by thioredoxin, has not been extensively studied. Selenoprotein thioredoxin reductase 1 (Txnrd1) is important for attenuating activation of the apoptosis signaling-regulating kinase 1 (ASK1) and the c-Jun N-terminal kinase (JNK) pathway caused by high doses of APAP. Therefore, a detailed investigation of the role of Txnrd1 in APAP-induced hepatotoxicity was conducted. Liver-specific Txnrd1 knockout mice (Txnrd1(ΔLiv)) were generated and treated with a hepatotoxic dose (400 mg/kg) of APAP for 1 or 6 h. Liver toxicity was assessed by measuring the activities of liver enzymes aspartate aminotransferase and alanine aminotransferase in serum, in addition to histopathological analysis of liver sections and analysis of glutathione levels. At 1 h post-APAP treatment, total and mitochondrial glutathione levels in control and Txnrd1(ΔLiv) mice were similarly depleted. However, at 6 h post-APAP treatment, Txnrd1(ΔLiv) mice were resistant to APAP toxicity as liver enzymes and histology were not significantly different from the corresponding untreated mice. Analyses revealed the compensatory up-regulation of many of the nuclear factor erythroid 2-related factor 2 (NRF2) target genes and proteins in Txnrd1(ΔLiv) mice with and without APAP treatment. Yet, JNK was phosphorylated to a similar extent in APAP-treated control mice. The results suggest that Txnrd1(ΔLiv) mice are primed for xenobiotic detoxication primarily through NRF2 activation.

Identifying xenobiotic metabolites with in silico prediction tools and LCMS suspect screening analysis.

Understanding the metabolic fate of a xenobiotic substance can help inform its potential health risks and allow for the identification of signature metabolites associated with exposure. The need to characterize metabolites of poorly studied or novel substances has shifted exposure studies towards non-targeted analysis (NTA), which often aims to profile many compounds within a sample using high-resolution liquid-chromatography mass-spectrometry (LCMS). Here we evaluate the suitability of suspect screening analysis (SSA) liquid-chromatography mass-spectrometry to inform xenobiotic chemical metabolism. Given a lack of knowledge of true metabolites for most chemicals, predictive tools were used to generate potential metabolites as suspect screening lists to guide the identification of selected xenobiotic substances and their associated metabolites. Thirty-three substances were selected to represent a diverse array of pharmaceutical, agrochemical, and industrial chemicals from Environmental Protection Agency's ToxCast chemical library. The compounds were incubated in a metabolically-active in vitro assay using primary hepatocytes and the resulting supernatant and lysate fractions were analyzed with high-resolution LCMS. Metabolites were simulated for each compound structure using software and then combined to serve as the suspect screening list. The exact masses of the predicted metabolites were then used to select LCMS features for fragmentation via tandem mass spectrometry (MS/MS). Of the starting chemicals, 12 were measured in at least one sample in either positive or negative ion mode and a subset of these were used to develop the analysis workflow. We implemented a screening level workflow for background subtraction and the incorporation of time-varying kinetics into the identification of likely metabolites. We used haloperidol as a case study to perform an in-depth analysis, which resulted in identifying five known metabolites and five molecular features that represent potential novel metabolites, two of which were assigned discrete structures based on in silico predictions. This workflow was applied to five additional test chemicals, and 15 molecular features were selected as either reported metabolites, predicted metabolites, or potential metabolites without a structural assignment. This study demonstrates that in some-but not all-cases, suspect screening analysis methods provide a means to rapidly identify and characterize metabolites of xenobiotic chemicals.

Considerations from an International Regulatory and Pharmaceutical Industry (IQ MPS Affiliate) Workshop on the Standardization of Complex In Vitro Models in Drug Development.

In May 2022, there is an International Regulatory and Pharmaceutical Industry (Innovation and Quality [IQ] Microphysiological Systems [MPS] Affiliate) Workshop on the standardization of complex in vitro models (CIVMs) in drug development. This manuscript summarizes the discussions and conclusions of this joint workshop organized and executed by the IQ MPS Affiliate and the United States Food and Drug Administration (FDA). A key objective of the workshop is to facilitate discussions around opportunities and/or needs for standardization of MPS and chart potential pathways to increase model utilization in the context of regulatory decision making. Participation in the workshop included 200 attendees from the FDA, IQ MPS Affiliate, and 26 global regulatory organizations and affiliated parties representing Europe, Japan, and Canada. It is agreed that understanding global perspectives regarding the readiness of CIVM/MPS models for regulatory decision making and potential pathways to gaining acceptance is useful to align on globally. The obstacles are currently too great to develop standards for every context of use (COU). Instead, it is suggested that a more tractable approach may be to think of broadly applicable standards that can be applied regardless of COU and/or organ system. Considerations and next steps for this effort are described.

Diurnal regulation of the early growth response 1 (Egr-1) protein expression by hepatocyte nuclear factor 4alpha (HNF4alpha) and small heterodimer partner (SHP) cross-talk in liver fibrosis.

Early growth response 1 (Egr-1) protein is a critical regulator of genes contributing to liver fibrosis; however, little is known about the upstream transcriptional factors that control its expression. Here we show that Egr-1 expression is tightly regulated by nuclear receptor signaling. Hepatocyte nuclear factor 4α (HNF4α) activated the Egr-1 promoter through three DR1 response elements as identified by trans-activation assays. Deletion of these response elements or knockdown of HNF4α using siRNA largely abrogated Egr-1 promoter activation. HNF4α activity, as well as its enrichment on the Egr-1 promoter, were markedly repressed by small heterodimer partner (SHP) co-expression. Egr-1 mRNA and protein were transiently induced by HNF4α. On the contrary, HNF4α siRNA reduced Egr-1 expression at both the mRNA and protein levels, and overexpression of SHP reversed these effects. Conversely, knockdown of SHP by siRNA elevated Egr-1 protein. Interestingly, Egr-1 mRNA exhibited diurnal fluctuation, which was synchronized to the cyclic expression of SHP and HNF4α after cells were released from serum shock. Unexpectedly, the levels of Egr-1 mRNA and protein were highly up-regulated in Hnf4α(-/-) mice. Both HNF4α and Egr-1 expression were dramatically increased in SHP(-/-) mice with bile duct ligation and in human cirrhotic livers, which was inversely correlated with diminished SHP expression. In conclusion, our study revealed control network for Egr-1 expression through a feedback loop between SHP and HNF4α.

Metabolomics reveals attenuation of the SLC6A20 kidney transporter in nonhuman primate and mouse models of type 2 diabetes mellitus.

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na(+)-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.

The stable repression of mesenchymal program is required for hepatocyte identity: a novel role for hepatocyte nuclear factor 4α.

UNLABELLED: The concept that cellular terminal differentiation is stably maintained once development is complete has been questioned by numerous observations showing that differentiated epithelium may undergo an epithelial-to-mesenchymal transition (EMT) program. EMT and the reverse process, mesenchymal-to-epithelial transition (MET), are typical events of development, tissue repair, and tumor progression. In this study, we aimed to clarify the molecular mechanisms underlying these phenotypic conversions in hepatocytes. Hepatocyte nuclear factor 4α (HNF4α) was overexpressed in different hepatocyte cell lines and the resulting gene expression profile was determined by real-time quantitative polymerase chain reaction. HNF4α recruitment on promoters of both mesenchymal and EMT regulator genes was determined by way of electrophoretic mobility shift assay and chromatin immunoprecipitation. The effect of HNF4α depletion was assessed in silenced cells and in the context of the whole liver of HNF4 knockout animals. Our results identified key EMT regulators and mesenchymal genes as new targets of HNF4α. HNF4α, in cooperation with its target HNF1α, directly inhibits transcription of the EMT master regulatory genes Snail, Slug, and HMGA2 and of several mesenchymal markers. HNF4α-mediated repression of EMT genes induces MET in hepatomas, and its silencing triggers the mesenchymal program in differentiated hepatocytes both in cell culture and in the whole liver. CONCLUSION: The pivotal role of HNF4α in the induction and maintenance of hepatocyte differentiation should also be ascribed to its capacity to continuously repress the mesenchymal program; thus, both HNF4α activator and repressor functions are necessary for the identity of hepatocytes.

The expression of Phase II drug-metabolizing enzymes in human B-lymphoblastoid TK6 cells.

In vitro genotoxicity testing plays an important role in chemical risk assessment. The human B-lymphoblastoid cell line TK6 is widely used as a standard cell line for regulatory safety evaluations. Like many other mammalian cell lines, TK6 cells have limited metabolic capacity; therefore, usually require a source of exogenous metabolic activation for use in genotoxicity testing. Previously, we developed a set of TK6-derived cell lines that individually express one of fourteen cytochrome P450s (CYPs). In the present study, we surveyed a panel of major Phase II drug-metabolizing enzymes to characterize their baseline expression in TK6 cells. These results may serve as a reference enzymatic profile of this commonly used cell line.

High-throughput toxicogenomic screening of chemicals in the environment using metabolically competent hepatic cell cultures.

The ToxCast in vitro screening program has provided concentration-response bioactivity data across more than a thousand assay endpoints for thousands of chemicals found in our environment and commerce. However, most ToxCast screening assays have evaluated individual biological targets in cancer cell lines lacking integrated physiological functionality (such as receptor signaling, metabolism). We evaluated differentiated HepaRGTM cells, a human liver-derived cell model understood to effectively model physiologically relevant hepatic signaling. Expression of 93 gene transcripts was measured by quantitative polymerase chain reaction using Fluidigm 96.96 dynamic arrays in response to 1060 chemicals tested in eight-point concentration-response. A Bayesian framework quantitatively modeled chemical-induced changes in gene expression via six transcription factors including: aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, farnesoid X receptor, androgen receptor, and peroxisome proliferator-activated receptor alpha. For these chemicals the network model translates transcriptomic data into Bayesian inferences about molecular targets known to activate toxicological adverse outcome pathways. These data also provide new insights into the molecular signaling network of HepaRGTM cell cultures.

Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to PPARA agonism and fasting.

Exploring the molecular mechanisms that prevent inflammation during caloric restriction may yield promising therapeutic targets. During fasting, activation of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) promotes the utilization of lipids as an energy source. Herein, we show that ligand activation of PPARα directly upregulates the long non-coding RNA gene Gm15441 through PPARα binding sites within its promoter. Gm15441 expression suppresses its antisense transcript, encoding thioredoxin interacting protein (TXNIP). This, in turn, decreases TXNIP-stimulated NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, caspase-1 (CASP1) cleavage, and proinflammatory interleukin 1ß (IL1B) maturation. Gm15441-null mice were developed and shown to be more susceptible to NLRP3 inflammasome activation and to exhibit elevated CASP1 and IL1B cleavage in response to PPARα agonism and fasting. These findings provide evidence for a mechanism by which PPARα attenuates hepatic inflammasome activation in response to metabolic stress through induction of lncRNA Gm15441.

The role of Ah receptor in induction of human UDP-glucuronosyltransferase 1A1.

UDP-glucuronosyltransferases (UGTs) catalyze a major metabolic pathway initiating the transfer of glucuronic acid from uridine 5'-diphosphoglucuronic acid to endogenous and exogenous substances. Endogenous substances include bile acids, steroids, phenolic neurotransmitters, and bilirubin. Xenobiotic substances include dietary substances, therapeutics, and environmental compounds. The versatility in the selection of substrates for glucuronidation results from the multiplicity of the UGTs in addition to the ability of these genes to be regulated. UDP-glucuronosyltransferase 1A1 (UGT1A1), responsible for the glucuronidation of bilirubin, is controlled in a tissue-specific manner and can be regulated following environmental exposure. This chapter describes materials and methods for the examination of molecular interactions that control UGT1A1 expression and induction in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using an in vitro cell culture system, we mapped a regulatory sequence that contains a xenobiotic response element core sequence in the enhancer region of the UGT1A1 gene. Similar to regulation of CYP1A1, the transcriptional activation of UGT1A1 by TCDD is mediated through the aryl hydrocarbon receptor.

Metabolomics reveals a role for the chromatin-binding protein HMGN5 in glutathione metabolism.

High mobility group nucleosome-binding protein 5 (HMGN5) is a chromatin architectural protein that binds specifically to nucleosomes and reduces the compaction of the chromatin fiber. The protein is present in most vertebrate tissues however the physiological function of this protein is unknown. To examine the function of HMGN5 in vivo, mice lacking the nucleosome-binding domain of HMGN5 were generated and characterized. Serological analysis revealed that compared to wild-type littermates (Hmgn5(+/Y)), mice with a targeted mutation in the HMGN5 gene (Hmgn5(tm1/Y)), had elevated serum albumin, non-HDL cholesterol, triglycerides, and alanine transaminase, suggesting mild hepatic abnormalities. Metabolomics analysis of liver extracts and urine revealed clear differences in metabolites between Hmgn5(tm1/Y) and their Hmgn5(+/Y) littermates. Hmgn5(tm1/Y) mice had a significant increase in hepatic glutathione levels and decreased urinary concentrations of betaine, phenylacetylglycine, and creatine, all of which are metabolically related to the glutathione precursor glycine. Microarray and qPCR analysis revealed that expression of two genes affecting glutathione metabolism, glutathione peroxidase 6 (Gpx6) and hexokinase 1 (Hk1), was significantly decreased in Hmgn5(tm1/Y) mouse liver tissue. Analysis of chromatin structure by DNase I digestion revealed alterations in the chromatin structure of these genes in the livers of Hmgn5(tm1/Y) mice. Thus, functional loss of HMGN5 leads to changes in transcription of Gpx6 and Hk1 that alter glutathione metabolism.